Immunosuppressive effects of pteridine derivatives

ABSTRACT

This invention relates to a group of trisubstituted and tetrasubstituted pteridine derivatives, their pharmaceutically acceptable salts, N-oxides, solvates, dihydro- and tetrahydro-derivatives and enantiomers, possessing unexpectedly desirable pharmaceutical properties, in particular which are highly active immunosuppressive agents, and as such are useful in the treatment in transplant rejection and/or in the treatment of certain inflammatory diseases. These compounds are also useful in preventing or treating cardiovascular disorders, allergic conditions, disorders of the central nervous system and cell proliferative disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage of International ApplicationNo. PCT/BE2004/000124, filed Aug. 27, 2004, which, in turn, claims thebenefit of GB 0408955.3 filed on Apr. 22, 2004.

The invention relates to a class of novel pteridines. The inventionfurther relates to pharmaceutical compositions including a broad classof pteridines especially for the prevention and/or the treatment ofpathologic conditions such as, but not limited to, immune andauto-immune disorders, organ and cells transplant rejections, cellproliferative disorders, cardiovascular disorders, disorders of thecentral nervous system and viral diseases.

The invention further relates to combined pharmaceutical preparationscomprising one or more pteridines and one or more knownimmuno-suppressant drugs or antineoplastic drugs or anti-viral drugs.

This invention also relates to a method for the prevention and/ortreatment of pathologic conditions such as, but not limited to, immuneand autoimmune disorders, organ and cells transplant rejections, cellproliferative disorders, cardiovascular disorders, disorders of thecentral nervous system and viral diseases by the administration of aneffective amount of a specific pteridine optionally combined with one ormore known immunosuppressant drugs or antineoplastic drugs or anti-viraldrugs.

BACKGROUND OF THE INVENTION

Several 2,4-diaminopteridine derivatives being substituted in the6-position and/or the 7-position of the pteridine ring (according tostandard atom numbering for said ring) are known in the art, e.g. fromvarious sources of literature including Swiss Patent No. 231,852;British Patent No. 763,044; U.S. Pat. No. 2,512,572; U.S. Pat. No.2,581,889; U.S. Pat. No. 2,665,275; U.S. Pat. No. 2,667,486; U.S. Pat.No. 2,940,972; U.S. Pat. No. 3,081,230 and U.S. Pat. No. 5,047,405. Someof these substituted 2,4-diaminopteridine derivatives were disclosed inrelationship with various medical uses, such as bacterial growthinhibitors, antineoplastic agents, anti-schisto-somiasis activity,coronary dilating activity, diuretic and hypotensive activity, andanti-amnesic activity. In particular, U.S. Pat. No. 2,940,972 andEP-A-362,645 disclose very specific 2,4-diaminopteridine derivativesbeing substituted by piperidinyl, morpholinyl or pyrrolidinyl in the7-position of the pteridine ring.

Specific 2-aminopteridine derivatives wherein the 4-position of thepteridine ring is substituted with an alkoxy group and the 6-position isalso substituted are also known in the art, although without any medicalutility. For instance, U.S. Pat. No. 2,740,784 discloses suchderivatives wherein the 6-substituent is an acetal; J. Chem. Soc. (1957)2146-2158 discloses 2-dimethylamino-4-ethoxy-6-phenylpteridine as acompound with a melting point of 200° C.; Am. Khim. J. discloses2-amino-4-ethoxy-6,7-diphenylpteridine; Helv. Chem. Acta (1992)75:2317-2326 discloses 2-amino-4-pentoxy-6-methylthio-pteridine.Furthermore, International Patent Application published as WO 01/19825discloses 2-phenylamino and 2-phenylsulfoxide pteridines wherein the7-position is substituted with an amide as being useful inhibitors ofcell cycle regulatory kinases.

Nevertheless, there still is a need in the art for specific and highlytherapeutically active compounds, such as, but not limited to, drugs fortreating immune and autoimmune disorders, organ and cells transplantrejections, cell proliferative disorders, cardiovascular disorders,disorders of the central nervous system and viral diseases. Inparticular, there is a need in the art to provide immunosuppressivecompounds or antineoplastic drugs or anti-viral drugs which are activein a minor dose in order to replace existing drugs having significantside effects and to decrease treatment costs.

Currently used immunosuppressive drugs include antiproliferative agents,such as methotrexate (a 2,4-diaminopteridine derivative disclosed byU.S. Pat. No. 2,512,572), azathioprine, and cyclophosphamide. Sincethese drugs affect mitosis and cell division, they have severe toxiceffects on normal cells with high turn-over rate such as bone marrowcells and the gastrointestinal tract lining. Accordingly, marrowdepression and liver damage are common side effects of theseantiproliferative drugs.

Anti-inflammatory compounds used to induce immunosuppression includeadrenocortical steroids such as dexamethasone and prednisolone. Thecommon side effects observed with the use of these compounds arefrequent infections, abnormal metabolism, hypertension, and diabetes.

Other immunosuppressive compounds currently used to inhibit lymphocyteactivation and subsequent proliferation include cyclosporine, tacrolimusand rapamycin. Cyclosporine and its relatives are among the mostcommonly used immunosuppressant drugs. Cyclosporine is typically usedfor preventing or treating organ rejection in kidney, liver, heart,pancreas, bone marrow, and heart-lung transplants, as well as for thetreatment of autoimmune and inflammatory diseases such as Crohn'sdisease, aplastic anemia, multiple-sclerosis, myasthenia gravis,uveitis, biliary cirrhosis, etc. However, cyclosporines suffer from asmall therapeutic dose window and severe toxic effects includingnephrotoxicity, hepatotoxicity, hypertension, hirsutism, cancer, andneurotoxicity.

Additionally, monoclonal antibodies with immunosuppressant properties,such as OKT3, have been used to prevent and/or treat graft rejection.Introduction of such monoclonal antibodies into a patient, as with manybiological materials, induces several side-effects, such as dyspnea.Within the context of many life-threatening diseases, organtransplantation is considered a standard treatment and, in many cases,the only alternative to death. The immune response to foreign cellsurface antigens on the graft, encoded by the major histocompatibilitycomplex (hereinafter referred as MHC) and present on all cells,generally precludes successful transplantation of tissues and organsunless the transplant tissues come from a compatible donor and thenormal immune response is suppressed. Other than identical twins, thebest compatibility and thus, long term rates of engraftment, areachieved using MHC identical sibling donors or MHC identical unrelatedcadaver donors. However, such ideal matches are difficult to achieve.Further, with the increasing need of donor organs an increasing shortageof transplanted organs currently exists. Accordingly,xenotransplantation has emerged as an area of intensive study, but facesmany hurdles with regard to rejection within the recipient organism.

The host response to an organ allograft involves a complex series ofcellular interactions among T and B lymphocytes as well as macrophagesor dendritic cells that recognize and are activated by foreign antigen.Co-stimulatory factors, primarily cytokines, and specific cell-cellinteractions, provided by activated accessory cells such as macrophagesor dendritic cells are essential for T-cell proliferation. Thesemacrophages and dendritic cells either directly adhere to T-cellsthrough specific adhesion proteins or secrete cytokines that stimulateT-cells, such as IL-12 and IL-15. Accessory cell-derived co-stimulatorysignals stimulate activation of Interleukin-2 (IL-2) gene transcriptionand expression of high affinity IL-2 receptors in T-cells. IL-2 issecreted by T lymphocytes upon antigen stimulation and is required fornormal immune responsiveness. IL-2 stimulates lymphoid cells toproliferate and differentiate by binding to IL-2 specific cell surfacereceptors (IL-2R). IL-2 also initiates helper T-cell activation ofcytotoxic T-cells and stimulates secretion of interferon-γ which in turnactivates cytodestructive properties of macrophages. Furthermore, IFN-γand IL-4 are also important activators of MHC class II expression in thetransplanted organ, thereby further expanding the rejection cascade byenhancing the immunogenicity of the grafted organ The current model of aT-cell mediated response suggests that T-cells are primed in the T-cellzone of secondary lymphoid organs, primarily by dendritic cells. Theinitial interaction requires cell to cell contact between antigen-loadedMHC molecules on antigen-presenting cells (hereinafter referred as APC)and the T-cell receptor/CD3 complex on T-cells. Engagement of theTCR/CD3 complex induces CD154 expression predominantly on CD4 T-cellsthat in turn activate the APC through CD40 engagement, leading toimproved antigen presentation. This is caused partly by upregulation ofCD80 and CD86 expression on the APC, both of which are ligands for theimportant CD28 co-stimulatory molecule on T-cells. However, engagementof CD40 also leads to prolonged surface expression of MHC-antigencomplexes, expression of ligands for 4-1BB and OX-40 (potentco-stimulatory molecules expressed on activated T-cells). Furthermore,CD40 engagement leads to secretion of various cytokines (e.g., IL-12,IL-15, TNF-α, IL-1, IL-6, and IL-8) and chemokines, all of which haveimportant effects on both APC and T-cell activation and maturation.Similar mechanisms are involved in the development of auto-immunedisease, such as type I diabetes. In humans and non-obese diabetic mice,insulin-dependent diabetes mellitus results from a spontaneous T-celldependent auto-immune destruction of insulin-producing pancreatic .beta.cells that intensifies with age. The process is preceded by infiltrationof the islets with mononuclear cells (insulitis), primarily composed ofT lymphocytes. A delicate balance between auto-aggressive T-cells andsuppressor-type immune phenomena determines whether expression ofauto-immunity is limited to insulitis or not. Therapeutic strategiesthat target T-cells have been successful in preventing further progressof the auto-immune disease. These include neonatal thymectomy,administration of cyclosporine, and infusion of anti-pan T-cell,anti-CD4, or anti-CD25 (IL-2R) monoclonal antibodies. The aim of allrejection prevention and auto-immunity reversal strategies is tosuppress the patient's immune reactivity to the antigenic tissue oragent, with a minimum of morbidity and mortality. Accordingly, a numberof drugs are currently being used or investigated for theirimmunosuppressive properties. As discussed above, the most commonly usedimmunosuppressant is cyclosporine, which however has numerous sideeffects. Accordingly, in view of the relatively few choices for agentseffective at immunosuppression with low toxicity profiles and manageableside effects, there exists a need in the art for identification ofalternative immunosuppressive agents and for agents acting as complementto calcineurin inhibition.

The metastasis of cancer cells represents the primary source of clinicalmorbidity and mortality in the large majority of solid tumors.Metastasis of cancer cells may result from the entry of tumor cells intoeither lymphatic or blood vessels. Invasion of lymphatic vessels resultsin metastasis to regional draining lymph nodes. From the lymph nodes,melanoma cells for example tend to metastasize to the lung, liver, andbrain. For several solid tumors, including melanoma, the absence or thepresence of lymph nodes metastasis is the best predictor of patientsurvival. Presently, to our knowledge, no treatment is capable ofpreventing or significantly reducing metastasis. Hence, there is a needin the art for compounds having such anti-metastasis effect for asuitable treatment of cancer patients.

In the field of allergy, IgE is well known for inducing allergy mainlyby stimulating mast cells to release histamine. Also, asthma, beingcharacterized by inflammation of airway and bronchospasm, is mainlyinduced by Th2 cytokines such as IL-5, IL-10 or IL-13. Therefore thereis a need in the art for compounds that efficiently inhibit the releaseof these Th2 cytokines.

There is also a need in the art to improve therapeutic efficiency byproviding pharmaceutical compositions or combined preparationsexhibiting a synergistic effect as a result of combining two or moreimmunosuppressant drugs, or antineoplastic drugs or anti-viral drugs oranti-histamine drugs.

Meeting these various needs in the art constitutes the main goal of thepresent invention.

SUMMARY OF THE INVENTION

In a first embodiment, the present invention relates to a group of novelpteridine derivatives having the general formula (I):

wherein X represents an oxygen atom or a group with the formula S(O)_(m)wherein m is an integer from 0 to 2, or a group with the formula NZ andwherein:

-   -   R₁ is a group selected from the group consisting of C₁₋₇ alkyl,        C₂₋₇ alkenyl, C₂₋₇ alkynyl, C₃₋₁₀ cycloalkyl, C₃₋₁₀        cycloalkenyl, aryl, alkylaryl, arylalkyl, heterocyclic,        heterocyclic-substituted alkyl and alkyl-substituted        heterocyclic, each of said groups being optionally substituted        with one or more substituents selected from the group consisting        of halogen, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₂₋₇ alkenyl, C₂₋₇ alkynyl,        halo C₁₋₄ alkyl, C₃₋₁₀ cycloalkoxy, aryloxy, arylalkyloxy,        oxyheterocyclic, heterocyclic-substituted alkyloxy, thio C₁₋₇        alkyl, thio C₃₋₁₀ cycloalkyl, thioaryl, thioheterocyclic,        arylalkylthio, heterocyclic-substituted alkylthio, formyl,        hydroxyl, sulfhydryl, nitro, hydroxylamino, mercaptoamino,        cyano, carboxylic acid or esters or thioesters or amides or        thioamides or halides or anhydrides thereof, thiocarboxylic acid        or esters or thioesters or amides or thioamides or halides or        anhydrides thereof, carbamoyl, thiocarbamoyl, ureido,        thio-ureido, amino, cycloalkylamino, alkenylamino,        cycloalkenylamino, alkynylamino, arylamino, arylalkyl-amino,        hydroxylalkylamino, mercaptoalkyl-amino, heterocyclic amino,        hydrazino, alkylhydrazino and phenyl-hydrazino; or R₁ is a        carboxyalkyl, carboxyaryl, thiocarboxyaryl or thiocarboxyalkyl        group;    -   Z is a group independently defined as R₁ or Z is hydrogen or the        group NZ together with R₁ is either hydroxylamino or an        optionally substituted heterocyclic group containing at least        one nitrogen atom;    -   R₂ is selected from the group consisting of amino; acylamino;        thioacylamino; carbamoyl; thiocarbamoyl, ureido; thio-ureido,        sulfonamido; hydroxylamino; alkoxyamino; thioalkylamino;        mercaptoamino, hydrazino; alkylhydrazino; phenylhydrazino;        optionally substituted heterocyclic radicals; C₃₋₇ alkylamino;        arylamino; arylalkyl-amino; cycloalkylamino; alkenylamino;        cycloalkenylamino; heterocyclic amino; hydroxyalkylamino;        mercaptoalkylamino; C₁₋₇ alkoxy; C₃₋₁₀ cycloalkoxy; thio C₁₋₇        alkyl; arylsulfoxide; arylsulfone; heterocyclic sulfoxide;        heterocyclic sulfone; thio C₃₋₁₀ cycloalkyl; aryloxy; arylthio;        arylalkyloxy; arylalkylthio; oxyheterocyclic and        thioheterocyclic radicals;    -   R₄ is an atom or a group selected from the group consisting of        hydrogen; halogen; C₁₋₇ alkyl; C₂₋₇ alkenyl; C₂₋₇ alkynyl; halo        C₁₋₇ alkyl; carboxy C₁₋₇ alkyl; carboxyaryl; C₁₋₇ alkoxy; C₃₋₁₀        cycloalkoxy; aryloxy; arylalkyloxy; oxyheterocyclic;        heterocyclic-substituted alkyloxy; thio C₁₋₇ alkyl; thio C₃₋₁₀        cycloalkyl; thioaryl; thioheterocyclic; arylalkylthio;        heterocyclic-substituted alkylthio; hydroxylamino;        mercapto-amino; acylamino; thio-acylamino; alkoxyamino;        thioalkylamino; acetal; thio-acetal; carboxylic acid; carboxylic        acid esters, thioesters, halides, anhydrides, amides and        thioamides; thiocarboxylic acid; thiocarboxylic acid esters,        thioesters, halides, anhydrides, amides and thioamides;        hydroxyl; sulfhydryl; nitro; cyano; carbamoyl; thiocarbamoyl,        ureido; thio-ureido; alkylamino; cycloalkylamino; alkenylamino;        cycloalkenylamino; alkynylamino; arylamino; arylalkylamino;        hydroxyalkylamino; mercaptoalkylamino; heterocyclic amino;        heterocyclic-substituted alkylamino; oximino; alkyloximino;        hydrazino; alkylhydrazino; phenylhydrazino; cysteinyl acid,        esters, thioesters, halides, anhydrides, amides and thioamides        thereof; phenyl substituted with one or more substituents        selected from the group consisting of C₁₋₇ alkyl, C₂₋₇ alkenyl,        C₂₋₇ alkynyl, halo C₁₋₇ alkyl, nitro, hydroxy, sulfhydryl,        amino, C₃₋₁₀ cycloalkoxy, aryloxy, arylalkyloxy,        oxyheterocyclic, heterocyclic-substituted alkyloxy, thio C₁₋₇        alkyl, thio C₃₋₁₀ cycloalkyl, thioaryl, thioheterocyclic,        arylalkylthio, heterocyclic-substituted alkylthio, formyl,        carbamoyl, thiocarbamoyl, ureido, thio-ureido, sulfonamido,        hydroxylamino, alkoxyamino, mercaptoamino, thioalkylamino,        acylamino, thioacylamino, cyano, carboxylic acid or esters or        thioesters or halides or anhydrides or amides thereof,        thiocarboxylic acid or esters or thioesters or halides or        anhydrides or amides thereof, alkylamino, cycloalkylamino,        alkenylamino, cycloalkenylamino, alkynylamino, arylamino,        arylalkylamino, hydroxyalkylamino, mercaptoalkylamino,        heterocyclic amino, hydrazino, alkylhydrazino and        phenylhydrazino; aryl groups other than phenyl, the said aryl        groups being optionally substituted with one or more        substituents selected from the group consisting of halogen, C₁₋₇        alkyl, C₁₋₇ alkoxy, C₂₋₇ alkenyl, C₂₋₇ alkynyl, halo C₁₋₇ alkyl,        nitro, hydroxyl, sulfhydryl, amino, C₃₋₁₀ cycloalkoxy, aryloxy,        arylalkyloxy, oxyheterocyclic, heterocyclic-substituted        alkyloxy, thio C₁₋₇ alkyl, thio C₃₋₁₀ cycloalkyl, thioaryl,        thioheterocyclic, arylalkylthio, heterocyclic-substituted        alkylthio, formyl, carbamoyl, thiocarbamoyl, ureido,        thio-ureido, sulfonamido, hydroxylamino, alkoxyamino,        mercaptoamino, thioalkylamino, acylamino, thioacylamino, cyano,        carboxylic acid or esters or thioesters or halides or anhydrides        or amides thereof, thiocarboxylic acid or esters or thioesters        or halides or anhydrides or amides thereof, alkylamino,        cycloalkylamino, alkenylamino, cycloalkenylamino, alkynylamino,        arylamino, arylalkylamino, hydroxyalkylamino,        mercaptoalkylamino, heterocyclic amino, hydrazino,        alkylhydrazino and phenylhydrazino; optionally substituted        heterocyclic radicals other than piperidinyl, morpholinyl or        pyrrolidinyl, i.e. preferably selected from the group consisting        of oxabicycloheptyl, azabenzimidazolyl, azacycloheptyl,        azacyclooctyl, azacyclononyl, azabicyclononyl, tetrahydrofuryl,        tetrahydropyranyl, tetrahydropyronyl, tetrahydroquinoleoinyl,        tetrahydrothienyl and dioxide thereof, dihydrothienyl dioxide,        dioxindolyl, dioxinyl, dioxenyl, dioxazinyl, thioxanyl,        thioxolyl, thio-urazolyl, thiotriazolyl, thio-pyranyl,        thiopyronyl, coumarinyl, quinoleinyl, oxyquinoleinyl,        quinuclidinyl, xanthinyl, dihydropyranyl, benzodihydrofuryl,        benzothiopyronyl, benzothiopyranyl, benzoxazinyl, benzoxazolyl,        benzodioxolyl, benzodioxanyl, benzothiadiazolyl,        benzotrioazinyl, benzothiazolyl, benzoxazolyl, phenothioxinyl,        phenothiazolyl, phenothienyl, phenopyronyl, phenoxazolyl,        pyridinyl, dihydropyridinyl, tetrahydro-pyridinyl, piperidinyl,        thiomorpholinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl,        tetrazinyl, triazolyl, benzotriazolyl, tetrazolyl, imidazolyl,        pyrazolyl, thiazolyl, thiadiazolyl, isothiazolyl, oxazolyl,        oxadiazolyl, pyrrolyl, furyl, dihydrofuryl, furoyl, hydantoinyl,        dioxolanyl, dioxolyl, dithianyl, dithienyl, dithiinyl, thienyl,        indolyl, indazolyl, benzofuryl, quinolyl, quinazolinyl,        quinoxalinyl, carbazolyl, phenoxazinyl, phenothiazinyl,        xanthenyl, purinyl, benzothienyl, naphtothienyl, thianthrenyl,        pyranyl, pyronyl, benzopyronyl, isobenzofuranyl, chromenyl,        phenoxathiinyl, indolizinyl, quinolizinyl, isoquinolyl,        phthalazinyl, naphthiridinyl, cinnolinyl, pteridinyl,        carbolinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl,        phenothiazinyl, imidazolinyl, imidazolidinyl, benzimidazolyl,        pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl,        piperazinyl, uridinyl, thymidinyl, cytidinyl, azirinyl,        aziridinyl, diazirinyl, diaziridinyl, oxiranyl, oxaziridinyl,        dioxiranyl, thiiranyl, azetyl, dihydroazetyl, azetidinyl,        oxetyl, oxetanyl, thietyl, thietanyl, diazabicyclooctyl,        diazetyl, diaziridinonyl, diaziridinethionyl, chromanyl,        chromanonyl, thiochromanyl, thiochromanonyl, thiochromenyl,        benzofuranyl, benzisothiazolyl, benzocarbazolyl, benzochromonyl,        benzisoalloxazinyl, benzocoumarinyl, thiocoumarinyl,        phenometoxazinyl, phenoparoxazinyl, phentriazinyl, thiodiazinyl,        thiodiazolyl, indoxyl, thioindoxyl, benzodiazinyl, phtalidyl,        phtalimidinyl, phtalazonyl, alloxazinyl, xanthionyl, isatyl,        isopyrazolyl, isopyrazolonyl, urazolyl, urazinyl, uretinyl,        uretidinyl, succinyl, succinimido, benzylsultimyl and        benzylsultamyl; aromatic or heterocyclic substituents        substituted with an aliphatic spacer between the pteridine ring        and the aromatic or heterocyclic substituent, whereby said        aliphatic spacer is a branched or straight, saturated or        unsaturated aliphatic chain of 1 to 4 carbon atoms which may        contain one or more functions, atoms or radicals selected from        the group consisting of carbonyl (oxo), thiocarbonyl, alcohol        (hydroxyl), thiol, ether, thioether, acetal, thioacetal, amino,        imino, oximino, alkyloximino, amino-acid, cyano, acylamino,        thioacylamino, carbamoyl, thiocarbamoyl, ureido, thioureido,        carboxylic acid or ester or thioester or halide or anhydride or        amide, thiocarboxylic acid or ester or thioester or halide or        anhydride or amide, nitro, thio C₁₋₇ alkyl, thio C₃₋₁₀        cycloalkyl, hydroxylamino, mercaptoamino, alkylamino,        cycloalkylamino, alkenylamino, cycloalkenylamino, alkynylamino,        arylamino, arylalkylamino, hydroxyalkylamino,        mercaptoalkylamino, heterocyclic amino, hydrazino,        alkylhydrazino, phenylhydrazino, sulfonyl, sulfinyl, sulfonamido        and halogen; branched or straight, saturated or unsaturated        aliphatic chains of 1 to 7 carbon atoms optionally containing        one or more functions selected from the group consisting of        carbonyl (oxo), thiocarbonyl, alcohol (hydroxyl), thiol, ether,        thioether, acetal, thioacetal, amino, imino, oximino,        alkyloximino, aminoacid, cyano, acylamino; thioacylamino;        carbamoyl, thiocarbamoyl, ureido, thioureido, carboxylic acid        ester or halide or anhydride or amide, thiocarboxylic acid or        ester or thioester or halide or anhydride or amide, nitro, thio        C₁₋₇ alkyl, thio C₃₋₁₀ cycloalkyl, hydroxylamino, mercaptoamino,        alkylamino, cycloalkylamino, alkenylamino, cycloalkenylamino,        alkynylamino, arylamino, arylalkylamino, hydroxyalkylamino,        mercaptoalkylamino, heterocyclic amino, hydrazino,        alkylhydrazino, phenylhydrazino, sulfonyl, sulfinyl, sulfonamido        and halogen; and    -   R₃ is an atom or a group selected from the group consisting of        fluoro, bromo, iodo, C₂₋₇ alkyl; C₂₋₇ alkenyl; C₂₋₇ alkynyl;        halo C₁₋₇ alkyl; C₁₋₇ alkoxy; C₃₋₁₀ cycloalkoxy; aryloxy;        arylalkyloxy; oxyheterocyclic; heterocyclic-substituted        alkyloxy; thio C₂₋₇ alkyl; thio C₃₋₁₀ cycloalkyl; thioaryl;        thioheterocyclic; arylalkylthio; heterocyclic-substituted        alkylthio; hydroxylamino; alkoxyamino; thioalkylamino;        mercaptoamino; acyl-amino; thio-acylamino; thio-acetal;        carboxylic acid; carboxylic acid esters, thioesters, amides,        halides, anhydrides and thioamides; thiocarboxylic acid;        thiocarboxylic acid esters, thioesters, amides, halides,        anhydrides and thioamides; hydroxyl; sulfhydryl; nitro;        carbamoyl; thiocarbamoyl; ureido; thio-ureido; amino;        alkylamino; cycloalkylamino; alkenylamino; cycloalkenylamino;        alkynylamino; arylamino; arylalkylamino; hydroxyalkylamino;        mercaptoalkylamino; heterocyclic amino; heterocyclic-substituted        alkylamino; oximino; alkyloximino; hydrazino; alkylhydrazino;        phenylhydrazino; cysteinyl acid, esters, thioesters, amides and        thioamides thereof; aryl optionally substituted with one or more        substituents selected from the group consisting of halogen, C₁₋₇        alkyl, C₁₋₇ alkoxy, C₂₋₇ alkenyl, C₂₋₇ alkynyl, halo C₁₋₇ alkyl,        nitro, hydroxyl, sulfhydryl, amino, C₃₋₁₀ cycloalkoxy, aryloxy,        arylalkyloxy, oxyheterocyclic, heterocyclic-substituted        alkyloxy, thio C₁₋₇ alkyl, thio C₃₋₁₀ cycloalkyl, thioaryl,        thioheterocyclic, arylalkylthio, heterocyclic-substituted        alkylthio, formyl, carbamoyl, thiocarbamoyl, ureido,        thio-ureido, sulfonamido, hydroxylamino, mercaptoamino,        alkoxyamino, thioalkylamino, acylamino, thioacylamino, cyano,        carboxylic acid or esters or thioesters or halides or anhydrides        or amides thereof, thiocarboxylic acid or esters or thioesters        or halides or anhydrides or amides thereof, alkylamino,        cycloalkylamino, alkenylamino, cycloalkenylamino, alkynylamino,        arylamino, arylalkylamino, hydroxylalkylamino,        mercaptoalkylamino, heterocyclic amino, hydrazino,        alkylhydrazino and phenylhydrazino; optionally substituted        heterocyclic radicals; aromatic or heterocyclic substituents        substituted with an aliphatic spacer between the pteridine ring        and the aromatic or heterocyclic substituent, whereby said        aliphatic spacer is a branched or straight, saturated or        unsaturated aliphatic chain of 1 to 4 carbon atoms which may        contain one or more functions, atoms or radicals selected from        the group consisting of carbonyl (oxo), thiocarbonyl, alcohol        (hydroxyl), thiol, ether, thio-ether, acetal, thio-acetal,        amino, imino, oximino, alkyloximino, amino-add, cyano,        carboxylic acid or ester or thioester or amide, nitro, thio C₁₋₇        alkyl, thio C₃₋₁₀ cycloalkyl, alkylamino, cycloalkylamino,        alkenylamino, cycloalkenylamino, alkynylamino, arylamino,        arylalkylamino, hydroxyalkyl-amino, mercaptoalkylamino,        heterocyclic amino, hydrazino, alkylhydrazino, phenylhydrazino,        sulfonyl, sulfonamido and halogen; branched or straight,        saturated or unsaturated aliphatic chains of 2 to 7 carbon atoms        optionally containing one or more functions selected from the        group consisting of thiocarbonyl, alcohol (hydroxyl), thiol,        ether, thioether, thioacetal, amino, imino, oximino,        alkyloximino, amino-acid, cyano, acylamino, thioacylamino,        carbamoyl, thiocarbamoyl, ureido, thioureido, carboxylic acid or        ester or thioester or halide or anhydride or amide, thio        carboxylic acid or ester or thioester or halide or anhydride or        amide, nitro, thio C₁₋₇ alkyl, thio C₃₋₁₀ cycloalkyl,        hydroxylamino, mercaptoamino, alkylamino, cycloalkylamino,        alkenylamino, cycloalkenylamino, alkynylamino, arylamino,        arylalkylamino, hydroxyalkylamino, mercaptoalkylamino,        heterocyclic amino, hydrazino, alkylhydrazino, phenylhydrazino,        sulfonyl, sulfinyl, sulfonamido and halogen; or R₃ together with        R₄ forms a homocyclic or heterocyclic radical such as, but not        limited to, indolyl, dihydroxypyrimidyl or tetramethylene;        and/or a pharmaceutically acceptable addition salt thereof        and/or a stereoisomer thereof and/or a mono- or a di-N-oxide        thereof and/or a solvate thereof and/or a dihydro- or        tetrahydropteridine derivative thereof.

The above novel compounds have in common the structural features presentin the general formula (I), in particular they are at leasttrisubstituted in positions 2, 4 and 6 of the pteridine ring. They alsohave a potential specific biological activity profile and consequentusefulness in medicinal chemistry.

In a second embodiment, the present invention relates to the unexpectedfinding that at least one desirable biological property such as, but notlimited to, the ability to decrease the proliferation of lymphocytes, orto decrease T-cell activation, or to decrease B-cell or monocytes ormacrophages activation, or to inhibit the release of certain cytokines,is a common feature which is not only present in the group of novelcompounds defined in the general formula (I), but also in a group ofpteridine derivatives which is broader than the said group of novelcompounds. As a consequence, the invention relates to pharmaceuticalcompositions comprising as an active principle at least one pteridinederivative having the general formula (II):

wherein X represents an oxygen atom or a group with the formula S(O)_(m)wherein m is an integer from 0 to 2, or a group with the formula NZ andwherein:

-   -   R₁ is a group selected from the group consisting of C₁₋₇ alkyl,        C₂₋₇ alkenyl, C₂₋₇ alkynyl, C₃₋₁₀ cycloalkyl, C₃₋₁₀        cycloalkenyl, aryl, alkylaryl, arylalkyl, heterocyclic,        heterocyclic-substituted alkyl and alkyl-substituted        heterocyclic, each of said groups being optionally substituted        with one or more substituents selected from the group consisting        of halogen, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₂₋₇alkenyl, C₂₋₇ alkynyl,        halo C₁₋₄ alkyl, C₃₋₁₀ cycloalkoxy, aryloxy, arylalkyloxy,        oxyheterocyclic, heterocyclic-substituted alkyloxy, thio C₁₋₇        alkyl, thio C₃₋₁₀ cycloalkyl, thioaryl, thioheterocyclic,        arylalkylthio, heterocyclic-substituted alkylthio, formyl,        hydroxyl, sulfhydryl, nitro, hydroxylamino, mercaptoamino,        cyano, carboxylic acid or esters or thioesters or amides or        thioamides or halides or anhydrides thereof, thiocarboxylic acid        or esters or thioesters or amides or thioamides or halides or        anhydrides thereof, carbamoyl, thiocarbamoyl, ureido,        thio-ureido, amino, cycloalkylamino, alkenylamino,        cycloalkenylamino, alkynylamino, arylamino, arylalkyl-amino,        hydroxylalkylamino, mercaptoalkyl-amino, heterocyclic amino,        hydrazino, alkylhydrazino and phenyl-hydrazino; or R₁ is a        carboxyalkyl, carboxyaryl, thiocarboxyaryl or thiocarboxyalkyl        group;    -   Z is a group independently defined as R₁ or Z is hydrogen or the        group NZ together with R₁ is either hydroxylamino or an        optionally substituted heterocyclic group containing at least        one nitrogen atom;    -   R₂ is selected from the group consisting of amino; acylamino;        thioacylamino; carbamoyl; thiocarbamoyl, ureido; thioureido,        sulfon-amido; hydroxylamino; alkoxyamino; thioalkylamino;        mercaptoamino, hydrazino; alkylhydrazino; phenylhydrazino;        optionally substituted heterocyclic radicals; C₃₋₇ alkylamino;        arylamino; arylalkylamino; cycloalkylamino; alkenylamino;        cycloalkenylamino; heterocyclic amino; hydroxyalkylamino;        mercaptoalkylamino; C₁₋₇ alkoxy; C₃₋₁₀ cycloalkoxy; thio C₁₋₇        alkyl; arylsulfoxide; arylsulfone; heterocyclic sulfoxide;        heterocyclic sulfone; thio C₃₋₁₀ cycloalkyl; aryloxy; arylthio;        arylalkyloxy; arylalkylthio; oxyheterocyclic and        thioheterocyclic radicals,    -   R₄ is an atom or a group selected from the group consisting of        hydrogen; halogen; C₁₋₇ alkyl; C₂₋₇ alkenyl; C₂₋₇ alkynyl; halo        C₁₋₇ alkyl; carboxy C₁₋₇ alkyl; acetoxy C₁₋₇ alkyl; carboxyaryl;        C₁₋₇ alkoxy; C₃₋₁₀ cycloalkoxy; aryloxy; arylalkyloxy;        oxyheterocyclic; heterocyclic-substituted alkyloxy; thio C₁₋₇        alkyl; thio C₃₋₁₀ cycloalkyl; thioaryl; thioheterocyclic;        arylalkylthio; heterocyclic-substituted alkylthio;        hydroxylamino; mercapto-amino; acylamino; thio-acylamino;        alkoxyamino; thioalkylamino; acetal; thio-acetal; carboxylic        acid; carboxylic acid esters, thioesters, halides, anhydrides,        amides and thioamides; thiocarboxylic acid; thiocarboxylic acid        esters, thioesters, halides, anhydrides, amides and thioamides;        hydroxyl; sulfhydryl; nitro; cyano; carbamoyl; thiocarbamoyl,        ureido; thio-ureido; alkylamino; cycloalkylamino; alkenylamino;        cycloalkenylamino; alkynylamino; arylamino; arylalkylamino;        hydroxyalkylamino; mercaptoalkyl amino; heterocyclic amino;        heterocyclic-substituted alkylamino; oximino; alkyloximino;        hydrazino; alkylhydrazino; phenylhydrazino; cysteinyl acid,        esters, thioesters, halides, anhydrides, amides and thioamides        thereof; phenyl substituted with one or more substituents        selected from the group consisting of C₁₋₇ alkyl, C₂₋₇ alkenyl,        C₂₋₇ alkynyl, halo C₁₋₇ alkyl, nitro, hydroxyl, sulfhydryl,        amino, C₃₋₁₀ cycloalkoxy, aryloxy, arylalkyloxy,        oxyheterocyclic, heterocyclic-substituted alkyloxy, thio C₁₋₇        alkyl, thio C₃₋₁₀ cycloalkyl, thioaryl, thioheterocyclic,        arylalkylthio, heterocyclic-substituted alkylthio, formyl,        carbamoyl, thiocarbamoyl, ureido, thio-ureido, sulfonamido,        hydroxylamino, alkoxyamino, mercaptoamino, thioalkylamino,        acylamino, thioacylamino, cyano, carboxylic acid or esters or        thioesters or halides or anhydrides or amides thereof,        thiocarboxylic acid or esters or thioesters or halides or        anhydrides or amides thereof, alkylamino, cycloalkylamino,        alkenyl-amino, cycloalkenylamino, alkynylamino, arylamino,        arylalkylamino, hydroxyalkylamino, mercaptoalkylamino,        heterocyclic amino, hydrazino, alkylhydrazino and        phenylhydrazino; aryl groups other than phenyl, the said aryl        groups being optionally substituted with one or more        substituents selected from the group consisting of halogen, C₁₋₇        alkyl, C₁₋₇ alkoxy, C₂₋₇ alkenyl, C₂₋₇ alkynyl, halo C₁₋₇ alkyl,        nitro, hydroxyl, sulfhydryl, amino, C₃₋₁₀ cycloalkoxy, aryloxy,        arylalkyloxy, oxyhetero-cyclic, heterocyclic-substituted        alkyloxy, thio C₁₋₇ alkyl, thio C₃₋₁₀ cycloalkyl, thioaryl,        thioheterocyclic, arylalkylthio, heterocyclic-substituted        alkylthio, formyl, carbamoyl, thiocarbamoyl, ureido,        thio-ureido, sulfonamido, hydroxylamino, alkoxyamino,        mercaptoamino, thioalkylamino, acylamino, thioacylamino, cyano,        carboxylic acid or esters or thioesters or halides or anhydrides        or amides thereof, thiocarboxylic acid or esters or thioesters        or halides or anhydrides or amides thereof, alkylamino,        cycloalkylamino, alkenylamino, cyclo-alkenylamino, alkynylamino,        arylamino, arylalkylamino, hydroxyalkyl-amino,        mercaptoalkylamino, heterocyclic amino, hydrazino,        alkyl-hydrazino and phenylhydrazino; optionally substituted        heterocyclic radicals other than piperidinyl, morpholinyl or        pyrrolidinyl, i.e. preferably selected from the group consisting        of oxabicycloheptyl, azabenzimi-dazolyl, azacycloheptyl,        azacyclooctyl, azacyclononyl, azabicyclononyl, tetrahydrofuryl,        tetrahydropyranyl, tetrahydropyronyl, tetrahydroquino-leinyl,        tetrahydrothienyl and dioxide thereof, dihydrothienyl dioxide,        dioxindolyl, dioxinyl, dioxenyl, dioxazinyl, thioxanyl,        thioxolyl, thio-urazolyl, thiotriazolyl, thiopyranyl,        thiopyronyl, coumarinyl, quinoleinyl, oxyquinoleinyl,        quinuclidinyl, xanthinyl, dihydropyranyl, benzodihydro-furyl,        benzothiopyronyl, benzothiopyranyl, benzoxazinyl, benzoxazolyl,        benzodioxolyl, benzodioxanyl, benzothiadiazolyl, benzotriazinyl,        benzothiazolyl, benzoxazolyl, phenothioxinyl, phenothiazolyl,        phenothienyl, phenopyronyl, phenoxazolyl, pyridinyl,        dihydropyridinyl, tetrahydropyridinyl, piperidinyl,        thiomorpholinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl,        tetrazinyl, triazolyl, benzotriazolyl, tetrazolyl, imidazolyl,        pyrazolyl, thiazolyl, thiadiazolyl, isothiazolyl, oxazolyl,        oxadiazolyl, pyrrolyl, furyl, dihydrofuryl, furoyl, hydantoinyl,        dioxolanyl, dioxolyl, dithianyl, dithienyl, dithiinyl, thienyl,        indolyl, indazolyl, benzofuryl, quinolyl, quinazolinyl,        quinoxalinyl, carbazolyl, phenoxazinyl, phenothiazinyl,        xanthenyl, purinyl, benzothienyl, naphtothienyl, thianthrenyl,        pyranyl, pyronyl, benzopyronyl, isobenzo-furanyl, chromenyl,        phenoxathiinyl, indolizinyl, quinolizinyl, isoquinolyl,        phthalazinyl, naphthiridinyl, cinnolinyl, pteridinyl,        carbolinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl,        phenothiazinyl, imidazolinyl, imidazolidinyl, benzimidazolyl,        pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl,        piperazinyl, uridinyl, thymidinyl, cytidinyl, azirinyl,        aziridinyl, diazirinyl, diaziridinyl, oxiranyl, oxaziridinyl,        dioxiranyl, thiiranyl, azetyl, dihydroazetyl, azetidinyl,        oxetyl, oxetanyl, thietyl, thietanyl, diaza-bicyclooctyl,        diazetyl, diaziridinonyl, diaziridinethionyl, chromanyl,        chromanonyl, thiochromanyl, thiochromanonyl, thiochromenyl,        benzofuranyl, benzisothiazolyl, benzocarbazolyl, benzochromonyl,        benziso-alloxazinyl, benzocoumarinyl, thiocoumarinyl,        phenometoxa-zinyl, phenoparoxazinyl, phentriazinyl,        thiodiazinyl, thiodiazolyl, indoxyl, thioindoxyl, benzodiazinyl,        phtalidyl, phtalimidinyl, phtalazonyl, alloxazinyl, xanthionyl,        isatyl, isopyrazolyl, isopyrazolonyl, urazolyl, urazinyl,        uretinyl, uretidinyl, succinyl, succinimido, benzylsultimyl and        benzylsultamyl; aromatic or heterocyclic substituents        substituted with an aliphatic spacer between the pteridine ring        and the aromatic or heterocyclic substituent, whereby said        aliphatic spacer is a branched or straight, saturated or        unsaturated aliphatic chain of 1 to 4 carbon atoms which may        contain one or more functions, atoms or radicals selected from        the group consisting of carbonyl (oxo), thiocarbonyl, alcohol        (hydroxyl), thiol, ether, thio-ether, acetal, thio-acetal,        amino, imino, oximino, alkyloximino, amino-acid, cyano,        acylamino, thioacylamino, carbamoyl, thiocarbamoyl, ureido,        thio-ureido, carboxylic acid or ester or thioester or halide or        anhydride or amide, thiocarboxylic acid or ester or thioester or        halide or anhydride or amide, nitro, thio C₁₋₇ alkyl, thio C₃₋₁₀        cycloalkyl, hydroxylamino, mercaptoamino, alkylamino,        cycloalkyl-amino, alkenylamino, cycloalkenylamino, alkynylamino,        arylamino, arylalkylamino, hydroxyalkylamino,        mercaptoalkylamino, heterocyclic amino, hydrazino,        alkylhydrazino, phenylhydrazino, sulfonyl, sulfinyl, sulfonamido        and halogen; branched or straight, saturated or unsaturated        aliphatic chains of 2 to 7 carbon atoms optionally containing        one or more functions selected from the group consisting of        carbonyl (oxo), thiocarbonyl, alcohol (hydroxyl), thiol, ether,        thio-ether, acetal, thio-acetal, amino, imino, oximino,        alkyl-oximino, amino-acid, cyano, acylamino; thioacylamino;        carbamoyl, thiocarbamoyl, ureido, thio-ureido, carboxylic acid        ester or halide or anhydride or amide, thiocarboxylic acid or        ester or thioester or halide or anhydride or amide, nitro, thio        C₁₋₇ alkyl, thio C₃₋₁₀ cycloalkyl, hydroxylamino,        mercapto-amino, alkylamino, cycloalkylamino, alkenylamino,        cycloalkenylamino, alkynylamino, arylamino, arylalkylamino,        hydroxy-alkylamino, mercaptoalkylamino, heterocyclic amino,        hydrazino, alkylhydrazino, phenylhydrazino, sulfonyl, sulfinyl,        sulfonamido and halogen; and    -   R₃ is an atom or a group defined as R₄ or R₃ is selected from        the group consisting of morpholinyl, amino, hydrogen, methyl,        thiomethyl and chloro; or R₃ together with R₄ forms a homocyclic        or heterocyclic radical such as, but not limited to, indolyl,        dihydroxypyrimidyl or tetramethylene;        and/or a pharmaceutically acceptable addition salt thereof        and/or a stereoisomer thereof and/or a mono- or a di-N-oxide        thereof and/or a solvate and/or a dihydro- or        tetrahydropteridine derivative thereof.

Compounds of formula (II) are highly active immunosuppressive agents,antineoplastic agents, anti-allergic agents or anti-viral agents which,together with one or more pharmaceutically acceptable carriers, may beformulated into pharmaceutical compositions for the prevention ortreatment of pathologic conditions such as, but not limited to, immuneand autoimmune disorders, organ and cells transplant rejections,allergic conditions, cell proliferative disorders, cardiovasculardisorders, disorders of the central nervous system and viral diseases.

In a further embodiment, the present invention relates to combinedpreparations containing at least one compound of formula (II) and one ormore drugs such as immunosuppressant and/or immunomodulator drugs,antineoplastic drugs, anti-histamines, inhibitors of agents causative ofallergic conditions, or antiviral agents. In a further embodiment, thepresent invention relates to the prevention or treatment of theabove-cited pathologic conditions by administering to the patient inneed thereof an effective amount of a compound of general formula (II),optionally in the form of a pharmaceutical composition or combinedpreparation with another suitable drug.

In a still further embodiment, the present invention relates to variousprocesses and methods for making the novel pteridine derivatives definedin general formula (I), as well as their pharmaceutically acceptablesalts, N-oxides, solvates, enantiomers and dihydro- andtetrahydroderivatives.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents a scheme for the preparation of 2,4,6-trisubstitutedpteridine derivatives according to one embodiment of this invention,having various R₂ and R₃ substituents in the 2- and 6-positions of thepteridine ring, respectively.

FIGS. 2 to 5 represent alternative schemes for the preparation of 2,4,6-or 2,4,7-trisubstituted or 2,4,6,7-tetrasubstituted pteridinederivatives with various R₁, R₂, R₃ and/or R₄ substituents, according toother embodiments of this invention.

FIG. 6 represents a scheme for the preparation of symmetrical2,4,6-trisubstituted pteridines and 2,4,7-trisubstituted as well as2,4,6,7-tetrasubstituted pteridine derivatives according to anotherembodiment of this invention, i.e. wherein substituents in the 2- and4-positions of the pteridine ring are identical.

FIG. 7 represents a scheme for the preparation of 2,4,6-trisubstitutedpteridine derivatives according to one embodiment of this invention,wherein the substituent in the 6-position of the pteridine ring is aphenyl group substituted by a nitrogen-containing function.

FIG. 8 represents a scheme for the preparation of 2,4,6-trisubstitutedpteridine derivatives according to another embodiment of this invention,wherein the substituent in the 6-position of the pteridine ring is aphenyl group substituted by an oxygen-containing function.

DEFINITIONS

Unless otherwise stated herein, the term “trisubstituted” means thatthree of the carbon atoms being in positions 2, 4 and 6 or,alternatively, in positions 2, 4 and 7 of the pteridine ring (accordingto standard atom numbering for the pteridine ring) are substituted withan atom or group other than hydrogen. The term “tetrasubstituted” meansthat all four carbon atoms being in positions 2, 4, 6 and 7 of thepteridine ring are substituted with an atom or group other thanhydrogen.

As used herein with respect to a substituting radical, and unlessotherwise stated, the terms “C₁₋₇ alkyl” or “aliphatic saturatedhydrocarbon radicals with 1 to 7 carbon atoms” means straight andbranched chain saturated acyclic hydrocarbon monovalent radicals havingfrom 1 to 7 carbon atoms such as, for example, methyl, ethyl, propyl,n-butyl, 1-methylethyl (isopropyl), 2-methylpropyl(isobutyl),1,1-dimethylethyl(ter-butyl), 2-methylbutyl, n-pentyl, dimethylpropyl,n-hexyl, 2-methylpentyl, 3-methylpentyl, n-heptyl and the like; the term“C₁₋₄ alkyl” designate the corresponding radicals with only 1 to 4carbon atoms, and so on.

As used herein with respect to a substituting radical, and unlessotherwise stated, the term C₁₋₇ alkylene means the divalent hydrocarbonradical corresponding to the above defined C₁₋₇ alkyl, such asmethylene, bis(methylene), tris(methylene), tetramethylene,hexamethylene and the like.

As used herein with respect to a substituting radical, and unlessotherwise stated, the terms “C₃₋₁₀ cycloalkyl” and “cycloaliphaticsaturated hydrocarbon radical with 3 to 10 carbon atoms” means amonocyclic saturated hydrocarbon monovalent radical having from 3 to 10carbon atoms, such as for instance cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclooctyl and the like, or a C₇₋₁₀ polycyclicsaturated hydrocarbon monovalent radical having from 7 to 10 carbonatoms such as, for instance, norbornyl, fenchyl, trimethyltricycloheptylor adamantyl.

As used herein with respect to a substituting radical, and unlessotherwise stated, the term “C₃₋₁₀ cycloalkylene” means the divalenthydrocarbon radical corresponding to the above defined C₃₋₁₀ cycloalkyl.

As used herein with respect to a substituting radical, and unlessotherwise stated, the terms “aryl” and “aromatic substituent” areinterchangeable and designate any mono- or polyaromatic monovalenthydrocarbon radical having from 6 up to 30 carbon atoms such as but notlimited to phenyl, naphthyl, anthracenyl, adamantyl, phenantracyl,fluoranthenyl, chrysenyl, pyrenyl, biphenylyl, terphenyl, picenyl andthe like, including spiro hydrocarbon radicals and fused benzo —C₅₋₈cycloalkyl radicals (the latter being as defined above) such as, forinstance, indanyl, 1,2,3,4-tetrahydronaphtalenyl, fluorenyl and thelike.

As used herein with respect to a substituting radical such as thecombination of R₃ and R₄, and unless otherwise stated, the term“homo-cyclic” means a mono- or polycyclic, saturated or mono-unsaturatedor polyunsaturated hydrocarbon radical having from 4 up to 15 carbonatoms but including no heteroatom in the said ring.

As used herein with respect to a substituting radical, and unlessotherwise stated, the term “heterocyclic” means a mono- or polycyclic,saturated or mono-unsaturated or polyunsaturated monovalent hydrocarbonradical having from 2 up to 15 carbon atoms and including one or moreheteroatoms in a 3 to 10 membered ring (and optionally one or moreheteroatoms attached to one or more carbon atoms of said ring, forinstance in the form of a carbonyl or thiocarbonyl group) and/or to oneor more heteroatoms of said ring, for instance in the form of a sulfone,sulfoxide, N-oxide, phosphate, phosphonate or selenium oxide, each saidheteroatom being independently selected from the group consisting ofnitrogen, oxygen, sulfur, selenium and phosphorus, including benzo-fusedheterocyclic radicals, such as but not limited to oxabicycloheptyl,azabenzimidazolyl, azacycloheptyl, azacyclooctyl, azacyclononyl,azabicyclononyl, tetrahydrofuryl, tetrahydropyranyl, tetrahydropyronyl,tetrahydroquinoleinyl, tetrahydrothienyl and dioxide thereof,dihydrothienyl dioxide, dioxindolyl, dioxinyl, dioxenyl, dioxazinyl,thioxanyl, thioxolyl, thio-urazolyl, thiotriazolyl, thiopyranyl,thiopyronyl, coumarinyl, quinoleinyl, oxyquinoleinyl, quinuclidinyl,xanthinyl, dihydropyranyl, benzodihydrofuryl, benzothiopyronyl,benzothiopyranyl, benzoxazinyl, benzoxazolyl, benzodioxolyl,benzodioxanyl, benzothiadiazolyl, benzotriazinyl, benzothiazolyl,benzoxazolyl, phenothioxinyl, phenothiazolyl,phenothienyl(benzothiofuranyl), phenopyronyl, phenoxazolyl, pyridinyl,dihydropyridinyl, tetrahydropyridinyl, piperidinyl, morpholinyl,thiomorpholinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl,tetrazinyl, triazolyl, benzotriazolyl, tetrazolyl, imidazolyl,pyrazolyl, thiazolyl, thiadiazolyl, isothiazolyl, oxazolyl, oxadiazolyl,pyrrolyl, furyl, dihydrofuryl, furoyl, hydantoinyl, dioxolanyl,dioxolyl, dithianyl, dithienyl, dithiinyl, thienyl, indolyl, indazolyl,benzofuryl, quinolyl, quinazolinyl, quinoxalinyl, carbazolyl,phenoxazinyl, phenothiazinyl, xanthenyl, purinyl, benzothienyl,naphtothienyl, thianthrenyl, pyranyl, pyronyl, benzopyronyl,isobenzofuranyl, chromenyl, phenoxathiinyl, indolizinyl, quinolizinyl,isoquinolyl, phthalazinyl, naphthiridinyl, cinnolinyl, pteridinyl,carbolinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl,phenothiazinyl, imidazolinyl, imidazolidinyl, benzimidazolyl,pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, piperazinyl,uridinyl, thymidinyl, cytidinyl, azirinyl, aziridinyl, diazirinyl,diaziridinyl, oxiranyl, oxaziridinyl, dioxiranyl, thiiranyl, azetyl,dihydroazetyl, azetidinyl, oxetyl, oxetanyl, thietyl, thietanyl,diazabicyclo-octyl, diazetyl, diaziridinonyl, diaziridinethionyl,chromanyl, chromanonyl, thiochromanyl, thiochromanonyl, thiochromenyl,benzofuranyl, benzisothiazolyl, benzocarbazolyl, benzochromonyl,benzisoalloxazinyl, benzocoumarinyl, thiocoumarinyl, phenometoxazinyl,phenoparoxazinyl, phentriazinyl, thiodiazinyl, thiodiazolyl, indoxyl,thio-indoxyl, benzodiazinyl (e.g. phtalazinyl), phtalidyl,phtalimidinyl, phtalazonyl, alloxazinyl, dibenzopyronyl (i.e.xanthonyl), xanthionyl, isatyl, isopyrazolyl, isopyrazolonyl, urazolyl,urazinyl, uretinyl, uretidinyl, succinyl, succinimido, benzylsultimyl,benzylsultamyl and the like, including all possible isomeric formsthereof, wherein each carbon atom of the said ring may be substitutedwith a substituent selected from the group consisting of halogen, nitro,C₁₋₇ alkyl (optionally containing one or more functions or radicalsselected from the group consisting of carbonyl (oxo), alcohol(hydroxyl), ether (alkoxy), acetal, amino, imino, oximino, alkyloximino,amino-acid, cyano, carboxylic acid ester or amide, nitro, thio C₁₋₇alkyl, thio C₃₋₁₀ cycloalkyl, C₁₋₇ alkylamino, cycloalkylamino,alkenylamino, cycloalkenylamino, alkynylamino, arylamino,arylalkylamino, hydroxylalkylamino, mercapto-alkylamino, heterocyclicamino, hydrazino, alkylhydrazino, phenyl-hydrazino, sulfonyl,sulfonamido and halogen), C₃₋₇ alkenyl, C₂₋₇ alkynyl, halo C₁₋₇ alkyl,C₃₋₁₀ cycloalkyl, aryl, arylalkyl, alkylaryl, alkylacyl, arylacyl,hydroxyl, amino, C₁₋₇ alkylamino, cycloalkylamino, alkenylamino,cyclo-alkenylamino, alkynylamino, arylamino, arylalkylamino,hydroxyalkylamino, mercaptoalkylamino, hetero-cyclic amino, hydrazino,alkylhydrazino, phenylhydrazino, sulfhydryl, C₁₋₇ alkoxy, C₃₋₁₀cycloalkoxy, aryloxy, arylalkyloxy, oxyheterocyclic,heterocyclic-substituted alkyloxy, thio C₁₋₇ alkyl, thio C₃₋₁₀cycloalkyl, thioaryl, thioheterocyclic, arylalkylthio,heterocyclic-substituted alkylthio, formyl, hydroxylamino, cyano,carboxylic acid or esters or thioesters or amides thereof,thiocarboxylic acid or esters or thioesters or amides thereof; dependingupon the number of unsaturations in the 3 to 10 membered ring,heterocyclic radicals may be sub-divided into heteroaromatic (or“heteroaryl”) radicals and non-aromatic heterocyclic radicals; when aheteroatom of the said non-aromatic heterocyclic radical is nitrogen,the latter may be substituted with a substituent selected from the groupconsisting of C₁₋₇ alkyl, C₃₋₁₀ cycloalkyl, aryl, arylalkyl andalkylaryl.

As used herein with respect to a substituting radical, and unlessotherwise stated, the terms “C₁₋₇ alkoxy”, “C₃₋₁₀ cycloalkoxy”,“aryloxy”, “arylalkyloxy”, “oxyheterocyclic”, “thio C₁₋₇ alkyl”, “thioC₃₋₁₀ cycloalkyl”, “arylthio”, “arylalkylthio” and “thioheterocyclic”refer to substituents wherein a C₁₋₇ alkyl radical, respectively a C₃₋₁₀cycloalkyl, aryl, arylalkyl or heterocyclic radical (each of them suchas defined herein), are attached to an oxygen atom or a sulfur atomthrough a single bond, such as but not limited to methoxy, ethoxy,propoxy, butoxy, thioethyl, thiomethyl, phenyloxy, benzyloxy,mercaptobenzyl, cresoxy and the like.

As used herein with respect to a substituting atom, and unless otherwisestated, the term halogen means any atom selected from the groupconsisting of fluorine, chlorine, bromine and iodine.

As used herein with respect to a substituting radical, and unlessotherwise stated, the term “halo C₁₋₇ alkyl” means a C₁₋₇ alkyl radical(such as above defined) in which one or more hydrogen atoms areindependently replaced by one or more halogens (preferably fluorine,chlorine or bromine), such as but not limited to difluoromethyl,trifluoromethyl, trifluoroethyl, octafluoropentyl, dodecafluoroheptyl,dichloromethyl and the like; the term “halo C₁₋₄ alkyl” designate thecorresponding radical with only 1 to 4 carbon atoms, and so on.

As used herein with respect to a substituting radical, and unlessotherwise stated, the terms “C₂₋₇ alkenyl” and “aliphatic unsaturatedhydrocarbon radical with 2 to 7 carbon atoms” are interchangeable anddesignate a straight and branched acyclic hydrocarbon monovalent radicalhaving one or more ethylenical unsaturations and having from 2 to 7carbon atoms such as, for example, vinyl, 2-propenyl, 3-butenyl,2-butenyl, 2-pentenyl, 3-pentenyl, 3-methyl-2-butenyl, 3-hexenyl,2-hexenyl, 2-heptenyl, butadienyl, pentadienyl, hexadienyl, heptadienyl,heptatrienyl and the like, including all possible isomers thereof; theterm “C₃₋₇ alkenyl” designate the corresponding radical with only 3 to 7carbon atoms, and so on.

As used herein with respect to a substituting radical, and unlessotherwise stated, the terms “C₃₋₁₀ cycloalkenyl” and “cycloaliphaticunsaturated hydrocarbon radical with 3 to 10 carbon atoms” areinterchangeable and mean a monocyclic mono- or polyunsaturatedhydrocarbon monovalent radical having from 3 to 8 carbon atoms, such asfor instance cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl,cycloheptadienyl, cyclohepta-trienyl, cyclooctenyl, cyclooctadienyl andthe like, or a C₇₋₁₀ polycyclic mono- or polyunsaturated hydrocarbonmono-valent radical having from 7 to 10 carbon atoms such asdicyclopentadienyl, fenchenyl including all isomers thereof, such asα-pinolenyl), bicyclo[2.2.1]hept-2-enyl, bicyclo[2.2.1]hepta-2,5-dienyl,cyclo-fenchenyl and the like.

As used herein with respect to a substituting radical, and unlessotherwise stated, the term “C₂₋₇ alkynyl” defines straight and branchedchain hydrocarbon radicals containing one or more triple bonds andhaving from 2 to 20 carbon atoms such as, for example, acetylenyl,2-propynyl, 3-butynyl, 2-butynyl, 2-pentynyl, 3-pentynyl,3-methyl-2-butynyl, 3-hexynyl, 2-hexynyl and the like and all possibleisomers thereof.

As used herein with respect to a substituting radical, and unlessotherwise stated, the terms “arylalkyl” and “heterocyclic-substitutedalkyl” refer to an aliphatic saturated hydrocarbon monovalent radical,preferably a C₁₋₇ alkyl or a C₃₋₁₀ cycloalkyl such as defined above,onto which an aryl radical or respectively a heterocyclic radical (suchas defined above) is already bonded, such as but not limited to benzyl,pyridylmethyl, pyridylethyl, 2-(2-pyridyl)isopropyl, oxazolylbutyl,2-thienylmethyl and 2-furylmethyl.

As used herein with respect to a substituting radical, and unlessotherwise stated, the term “alkylaryl” and “alkyl-substitutedheterocyclic” refer to an aryl radical or respectively a heterocyclicradical (such as defined above) onto which is (are) already bonded oneor more aliphatic saturated hydrocarbon monovalent radicals, preferablyC₁₋₇ alkyl radicals or C₃₋₁₀ cycloalkyl radicals as defined above suchas, but not limited to, o-toluyl, m-toluyl, p-toluyl, mesityl and2,4,6-trimethylphenyl.

As used herein with respect to a substituting radical, and unlessotherwise stated, the terms “alkylamino”, “cycloalkylamino”,“alkenyl-amino”, “cycloalkenylamino”, “arylamino”, “arylalkylamino”,“heterocyclic amino”, “hydroxyalkylamino”, “mercaptoalkylamino” and“alkynylamino” mean that respectively one (thus monosubstituted amino)or even two (thus disubstituted amino) C₁₋₇ alkyl, C₃₋₁₀ cycloalkyl,C₂₋₇ alkenyl, C₃₋₁₀ cycloalkenyl, aryl, arylalkyl, heterocyclic, mono-or polyhydroxy C₁₋₇ alkyl, mono- or polymercapto C₁₋₇ alkyl or C₂₋₇alkynyl radical(s) (each of them as defined herein, respectively) is/areattached to a nitrogen atom through a single bond or, in the case ofheterocyclic, include a nitrogen atom, such as but not limited to,anilino, benzylamino, methylamino, dimethylamino, ethylamino,diethylamino, isopropylamino, propenylamino, n-butylamino,ter-butylamino, dibutylamino, morpholino-alkylamino, morpholinyl,piperidinyl, piperazinyl, hydroxymethylamino, β-hydroxyethylamino andethynylamino; this definition also includes mixed disubstituted aminoradicals wherein the nitrogen atom is attached to two such radicalsbelonging to two different sub-set of radicals, e.g. an alkyl radicaland an alkenyl radical, or to two different radicals within the samesub-set of radicals, e.g. methylethylamino; the term “C₃₋₇ alkyl-amino”designates the corresponding radical with only 3 to 7 carbon atoms inthe alkyl group(s) attached to nitrogen, for instance diisopropylamino,and so on; among disubstituted amino radicals, symetrically substitutedare usually preferred and more easily accessible.

As used herein with respect to a substituting radical, and unlessotherwise stated, the terms “(thio)carboxylic acid ester”,“(thio)carboxylic acid thioester” and “(thio)carboxylic acid amide”refer to radicals wherein the carboxyl or thiocarboxyl group is directlyattached to the pteridine ring (e.g. in the 6- and/or 7-position) andwherein said carboxyl or thiocarboxyl group is bonded to thehydrocarbonyl residue of an alcohol, a thiol, a polyol, a phenol, athiophenol, a primary or secondary amine, a polyamine, an amino-alcoholor ammonia, the said hydrocarbonyl residue being selected from the groupconsisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl,arylalkyl, alkylaryl, alkylamino, cycloalkylamino, alkenylamino,cycloalkenylamino, arylamino, arylalkylamino, heterocyclic amino,hydroxyalkylamino, mercapto-alkylamino or alkynylamino (such as abovedefined, respectively).

As used herein with respect to a substituting radical, and unlessotherwise stated, the term “amino-acid” refers to a radical derived froma molecule having the chemical formula H₂N—CHR—COOH, wherein R is theside group of atoms characterizing the amino-acid type; said moleculemay be one of the 20 naturally-occurring amino-acids or any similar nonnaturally-occurring amino-acid.

As used herein and unless otherwise stated, the term “stereoisomer”refers to all possible different isomeric as well as conformationalforms which the compounds of formula (I) or (II) may possess, inparticular all possible stereochemically and conformationally isomericforms, all diastereomers, enantiomers and/or conformers of the basicmolecular structure. Some compounds of the present invention may existin different tautomeric forms, all of the latter being included withinthe scope of the present invention.

As used herein and unless otherwise stated, the term “enantiomer” meanseach individual optically active form of a compound of the invention,having an optical purity or enantiomeric excess (as determined bymethods standard in the art) of at least 80% (i.e. at least 90% of oneenantiomer and at most 10% of the other enantiomer), preferably at least90% and more preferably at least 98%.

As used herein and unless otherwise stated, the term “solvate” includesany combination which may be formed by a pteridine derivative of thisinvention with a suitable inorganic solvent (e.g. hydrates) or organicsolvent, such as but not limited to alcohols, ketones, esters and thelike.

As used herein and unless otherwise stated, the terms “dihydro-pteridinederivative” and “tetrahydropteridine derivative” refer to thehydrogenation products of the pteridine derivatives having formula (I)or (II), i.e. derivatives wherein two hydrogen atoms are present inpositions 5 and 6, or 7 and 8, of the pteridine ring, or wherein fourhydrogen atoms are present in positions 5, 6, 7 and 8 of the said ring;such hydrogenated derivatives are easily accessible from the pteridinederivatives using hydrogenation methods well known in the art.

DETAILED DESCRIPTION OF THE INVENTION

A main object of the invention is to provide a pharmaceuticalcomposition having high immunosuppressive activity. Thus, the presentinvention relates in particular to the medical applications of a groupof pteridine derivatives, their pharmaceutically acceptable salts,N-oxides, solvates, dihydro- and tetrahydroderivatives and enantiomers,possessing unexpectedly desirable pharmaceutical properties, inparticular which are highly active immunosuppressive agents, and as suchare useful in the treatment in transplant rejection and/or in thetreatment of certain inflammatory diseases.

Surprisingly, the compounds of the present invention show a broadertherapeutic spectrum profile than merely immunosuppressive activity, asis evidenced by the results obtained in the diversity of test proceduresdisclosed hereinbelow. A further advantageous feature of the compoundsof the present invention resides in their excellent oral activity.

In the first embodiment of the invention, the novel pteridinederivatives are as defined in the general formula (I), wherein each ofthe substituents X, Z, R₁, R₂, R₃ and R₄ may correspond to any of thedefinitions given above (and, when X includes sulfur, wherein m may be0, 1 or 2), in particular with any of the individual meanings (such asillustrated above) of generic terms such as, but not limited to, “C₁₋₇alkyl”, “C₂₋₇ alkenyl”, “C₂₋₇alkynyl”, “aryl”, “alkylaryl”, “arylalkyl”,“alkylamino”, “cydoalkylamino”, “alkenylamino”, “alkynylamino”,“arylamino”, “arylalkylamino”, “C₁₋₇ alkoxy”, “C₃₋₁₀ cycloalkoxy”, “thioC₁₋₇ alkyl”, “thio C₃₋₁₀ cycloalkyl”, “halo C₁₋₇ alkyl”, “amino-acid”and the like.

When a mixture of enantiomers of a pteridine derivative having thegeneral formula (I) according to the invention is obtained duringsynthesis, the said mixture may be separated by means and methodsstandard in the art, e.g. liquid chromatography using one or moresuitable chiral stationary phases. The latter include, for example,polysaccharides, in particular cellulose or amylose derivatives.Commercially available polysaccharide-based chiral stationary phasessuitable for this purpose are ChiralCel™ CA, OA, OB, OC, OD, OF, OG, OJand OK, and Chiralpak™ AD, AS, OP(+) and OT(+). Appropriate eluents ormobile phases for use in combination with said polysaccharide-basedchiral stationary phases are hydrocarbons such as hexane and the like,optionally admixed with an alcohol such as ethanol, isopropanol and thelike. The above mixture of enantiomers may alternatively be separated byforming diastereoisomers, followed by separation of thediastereoisomers, e.g. by differential crystallization orchromatography. The resolving agent may be cleaved from the separateddiastereoisomers, e.g. by treatment with acids or bases, in order togenerate the pure enantiomers of the compounds of the invention.

Some preferred pteridine derivatives having the general formula (I) or(II) according to the Invention are more specifically Illustrated in thefollowing examples and defined in the following claims. For instance,useful pteridine species disclosed below include those wherein:

-   -   R₁ is selected from the group consisting of methyl, ethyl,        isopropyl, pentyl and benzyl, and/or    -   R₂ is amino, and/or    -   R₄ is hydrogen or methoxy, and/or    -   R₃ is 3-thienyl or a phenyl group with one or more substituents        (in the latter case, such substituents are preferably each        independently selected from the group consisting of fluoro,        methoxy, ethoxy, trifluoromethyl, dimethylamino, chloro, cyano,        methyl, ethyl, carboxymethyl, methylthio, dimethylcarboxamido,        diethylcarboxamido and methylcarboxylate, and/or    -   X is a sulfur atom (i.e. m is 0) or an oxygen atom, or    -   X is NZ, wherein Z is selected from the group consisting of        hydrogen, methyl, ethyl, isopropyl and benzyl, or NZ together        with R₁ forms a radical selected from the group consisting of        hydroxylamino, morpholinyl, piperidinyl, piperazinyl,        N-methylpiperazinyl, 1,2,4-triazolyl and pyrrolidinyl.

The present invention further provides processes and methods for makingthe novel pteridine derivatives having the general formula (I). As ageneral rule, the preparation of these compounds is based on theprinciple that, starting from a suitable pteridine precursor, each ofthe substituents XR₁, R₂, R₃ and R₄ may be introduced separately(except, of course, when R₃ together with R₄ forms a homocyclic orheterocyclic radical) without adversely influencing the presence of oneor more substituents already introduced at other positions on thepteridine ring or the capacity to introduce further substituents lateron.

Methods of manufacture have been developed by the present inventorswhich may be used alternatively to, or may be combined with, the methodsof synthesis already known in the art of pteridine derivatives(depending upon the targeted final compound). For instance, methods forsimultaneously introducing R₃ and R₄ in the form of a homocyclic orheterocyclic radical at positions 6 and 7 of the pteridine ring arealready known from U.S. Pat. No. 2,581,889. The synthesis of mono- anddi-N-oxides of the pteridine derivatives of this invention can easily beachieved by treating the said derivatives with an oxidizing agent suchas, but not limited to, hydrogen peroxide (e.g. in the presence ofacetic acid) or a peracid such as chloroperbenzoic acid. Dihydro- andtetrahydropteridine derivatives of this invention can easily be obtainedby catalytic hydrogenation of the corresponding pteridine derivatives,e.g. by placing the latter in a hydrogen atmosphere in the presence ofplatinum oxide or platinum. The methods for making the pteridinederivatives of the present invention will now be explained in moredetails by reference to the appended FIGS. 1 to 8 wherein, unlessotherwise stated hereinafter, each of the substituting groups or atomsX, Z, R₁, R₂, R₃ and R₄ is as defined in formula (I) of the summary ofthe invention and, more specifically, may correspond to any of theindividual meanings disclosed above. The same manufacturing methods mayalso be applied, if need be, while starting from pteridine derivativeswhich are already known in the art. In the description of the reactionsteps involved in each figure, reference is made to the use of certaincatalysts and/or certain types of solvents. It should be understood thateach catalyst mentioned should be used in a catalytic amount well knownto the skilled person with respect to the type of reaction involved.Solvents that may be used in the following reaction steps includevarious kinds of organic solvents such as protic solvents, polar aproticsolvents and non-polar solvents as well as aqueous solvents which areinert under the relevant reaction conditions. More specific examplesinclude aromatic hydrocarbons, chlorinated hydrocarbons, ethers,aliphatic hydrocarbons, alcohols, esters, ketones, amides, water ormixtures thereof, as well as supercritical solvents such as carbondioxide (while performing the reaction under supercritical conditions).The suitable reaction temperature and pressure conditions applicable toeach kind of reaction step will not be detailed herein but do not departfrom the relevant conditions already known to the skilled person withrespect to the type of reaction involved and the type of solvent used(in particular its boiling point).

FIG. 1 represents a scheme for the preparation of 2,4,6-trisubstitutedpteridines with various R₂ and R₃ substituents in the 2- and 6-positionsof the pteridine ring, respectively. In the first step (a), achloropyrimidine 1, wherein R₂ may be inter alia amino, alkylamino,arylamino, alkoxy, aryloxy, mercaptoalkyl, or mercaptoaryl, is reactedwith an appropriate nucleophile R₁XH, the said nucleophile beingselected from the group consisting of alcohols (e.g. methanol, ethanol,isopropanol or benzylalcohol), thiols, primary amines and secondaryamines wherein R₁ may be inter alia alkyl, cycloalkyl, aryl, alkylaryl,heteroaryl or alkylheteroaryl. Introduction of a nitroso group into thepyrimidine intermediate 2 occurs in step (b) under acidic aqueousconditions in the presence of sodium nitrite NaNO₂. Reduction of thenitroso functionality of the pyrimidine intermediate 3 into a free aminogroup in intermediate 4 is then effected in step (c) by means ofreducing agents (such as Na₂S₂O₄ or (NH₄)₂S) in water, or catalytically(Pt/H₂) in the presence of a protic solvent. In step (d), ring closureis performed by treating the diaminopyrimidine 4 with glyoxal in orderto form a pteridine ring. In step (e), the nitrogen atom at position 8of the pteridine ring of compound 5 is oxidized, e.g. using H₂O₂ underacidic conditions. In step (f), a chlorine atom is regioselectivelyintroduced on the 6 position of the pteridine ring of compound 6 bytreatment with a carboxylic acid choride such as acetyl chloride underacidic conditions. Then in step (g) the 6-chlorosubstituted pteridine 7is reacted with a boronic acid having the general formula R₃B(OH)₂,wherein R₃ may be alkyl, cycloalkyl, aryl or heteroaryl, under basicconditions (such as in the presence of an aqueous alcaline solution) anda palladium based catalyst, thus yielding the desired derivative 8 ofthe present invention.

FIG. 2 represents a scheme for the preparation of 2,4,6- or2,4,7-trisubstituted or 2,4,6,7-tetrasubstituted pteridine derivativeswith various R₁, R₂, R₃ and/or R₄ substituents. In step (a), the thiolfunction of 2-mercapto-4,6-diaminopyrimidine is alkylated, preferablymethylated by reaction with methyl iodide in the presence of a solventsuch as ethanol, in order to yield 2-thiomethyl-4,6-diaminopyrimidine.Introduction of a nitroso group in the 5-position of the pyrimidine ringis then achieved in step (b) by using sodium nitrite under aqueousacidic conditions. In step (c), the methylthio group in the 2-positionis exchanged for a group R₂ by reaction with an appropriate nucleophile,wherein R₂ is as defined above and preferably is primary or secondaryamino, C₁₋₇ alkoxy, aryloxy, C₃₋₁₀ cycloalkoxy, heteroaryloxy, mercaptoC₁₋₇alkyl, mercaptoaryl, mercapto C₃₋₁₀cycloalkyl ormercapto-heteroaryl. Reduction of the nitroso group is then achieved instep (d) either catalytically (Pt/H₂) in the presence of a proticsolvent or chemically using sodium dithionite or ammonium sulfide in thepresence of water. Then in step (e), the resulting2-R₂-substituted-4,5,6-triaminopyrimidine is condensed, under acidiccondi-tions in the presence of a solvent such as methanol, with anα-ketoaldoxime bearing the group R₃, wherein R₃ may be C₁₋₇ alkyl, C₃₋₁₀cycloalkyl, aryl or heteroaryl, into a 2,6-substituted-4-aminopteridinederivative. Alternatively, the corresponding2,7-substituted-4-aminopteridine derivative can be obtained in step (f)by reacting the 2-R₂-substituted-4,5,6-triaminopyrimidine with amonosubstituted glyoxal bearing a group R₄, wherein R₄ may be inter aliaC₁₋₇ alkyl, C₃₋₁₀ cycloalkyl, aryl or heteroaryl. Alternatively, a2-R₂-substituted 4-amino-6,7-disubstituted pteridine derivative can beobtained in step (g) by reacting the 2-R₂-substituted4,5,6-triaminopyrimidine with a disubstituted glyoxal bearing groups R₃and R₄, wherein each of R₃ and R₄ is independently selected (i.e. R₃ andR₄ may be identical or different) from the group consisting of C₁₋₇alkyl, C₃₋₁₀ cycloalkyl, aryl and heteroaryl, under neutral or basicconditions. In step (h), acidic or basic hydrolysis of the amino groupat position 4 of the pteridine ring is performed and results in thecorresponding 4-oxopteridine derivative. In step (i), the hydroxyl groupof the tautomeric form of the latter is activated by nucleophilicdisplacement, e.g. by preparing the 4-[(1,2,4)-triazolyl] pteridinederivative. Finally in a first part of step (j), a nucleophilicdisplacement is performed by mixing the said 4-triazolylpteridinederivative with a nucleophile having the general formula R₁XH, such asfor example a primary or secondary amine, C₁₋₇ alkoxy, aryloxy, C₃₋₁₀cycloalkoxy, heteroaryloxy, mercapto C₁₋₇ alkyl, mercaptoaryl, mercaptoC₃₋₁₀ cycloalkyl, or mercapto-heteroaryl yielding the desired finalpteridine derivatives.

FIG. 3 represents a scheme for the preparation of 2,4,6- or2,4,7-trisubstituted or 2,4,6,7-tetrasubstituted pteridine derivativeswith various substituents on the pteridine ring, starting from the2-thiomethyl-5-nitroso-4,6-diaminopyrimidine obtained after step (b) ofthe scheme shown in FIG. 2. Reduction of the nitroso group is achievedin step (a) either catalytically (Pt/H₂) in the presence of a proticsolvent or chemically using sodium dithionite or ammonium sulfide in thepresence of water. Then in step (b),2-thiomethyl-4,5,6-triaminopyrimidine is condensed, under acidicconditions in the presence of a solvent such as methanol, with anα-ketoaldoxime bearing the group R₃, wherein R₃ may be inter alia C₁₋₇alkyl, C₃₋₁₀ cycloalkyl, aryl or heteroaryl, thus regioselectivelyyielding a 2-thiomethyl-4-amino-6-R₃-substituted-pteridine derivative.Alternatively, the corresponding 2-thiomethyl-4-amino-7-R₄-substitutedpteridine is obtained in step (c) by reacting2-thiomethyl-4,5,6-triaminopyrimidine with a monosubstituted glyoxalbearing a group R₄, wherein R₄ may be inter alia C₁₋₇ alkyl, C₃₋₁₀cycloalkyl, aryl or heteroaryl. Alternatively, the corresponding2-thiomethyl-4-amino-6-R₃-7-R₄-substituted pteridine is obtained in step(d) by reacting 2-thiomethyl-4,5,6-triamino-pyrimidine with adisubstituted glyoxal bearing groups R₃ and R₄, wherein each of R₃ andR₄ is independently selected (i.e. R₃ and R₄ may be identical ordifferent) from the group consisting of C₁₋₇ alkyl, C₃₋₁₀ cycloalkyl,aryl and heteroaryl, under neutral or basic conditions. In step (e), themethylthio group in the 2-position is oxidized to the correspondingsulfone by using oxidizing agents such as chloroperoxybenzoic acid inchloroform or hydrogen peroxide in acetic acid. The methylsulfonyl groupis easily exchanged in step (f) by reaction with a nucleophile, such asfor example a primary or secondary amine, C₁₋₇ alkoxy, aryloxy, C₃₋₁₀cycloalkoxy, heteroaryloxy, mercapto C₁₋₇ alkyl, mercaptoaryl, mercaptoC₃₋₁₀ cycloalkyl, or mercapto-heteroaryl. In step (g), acidic or basichydrolysis of the amino group at position 4 of the pteridine ring isperformed and results in the corresponding 4-oxopteridine derivative. Instep (h), the hydroxyl group of the tautomeric form of the latter isactivated by nucleophilic displacement, e.g. by preparing the4-[(1,2,4)-triazolyl]pteridine derivative. In the last step (i), anucleophilic displacement is performed by mixing the said4-triazolylpteridine derivative with a nucleophile having the generalformula R₁XH, such as for example a primary or secondary amine, C₁₋₇alkoxy, aryloxy, C₃₋₁₀ cycloalkoxy, heteroaryloxy, mercapto C₁₋₇ alkyl,mercaptoaryl, mercapto C₃₋₁₀ cycloalkyl, or mercapto-heteroaryl.

FIG. 4 represents a scheme for the synthesis of unsymmetrical2,4,6-trisubstituted and 2,4,7-trisubstituted, as well as2,4,6,7-tetrasubstituted, pteridine derivatives with various R₁, R₂, R₃and/or R₄ substituents in the 2-, 4-, 6- and/or 7-positions of thepteridine ring, respectively. In step (a), a2-R₂-substituted-4,5,6-triamino-pyrimidine is condensed with anα-keto-aldoxime bearing a radical R₃, wherein R₃ may be inter aliaselected from the group consisting of C₁₋₇ alkyl, C₃₋₁₀ cycloalkyl, aryland heteroaryl, in a protic solvent such as methanol under acidicconditions yielding regioselectively a 4-amino-pteridine bearing aR₂-substituent in position 2 and a R₃ substituent in position 6 of thepteridine ring. Alternatively, a2-R₂-substituted-4-amino-7-R₄-substituted pteridine derivative can beobtained in step (b) by reacting a2-R₂-substituted-4,5,6-triamino-pyrimidine with a monosubstitutedglyoxal bearing the group R₄, wherein R₄ may be inter alia selected fromthe group consisting of C₁₋₇ alkyl, C₃₋₁₀ cycloalkyl, aryl andheteroaryl, under neutral or basic conditions. Alternatively, a2-R₂-substituted-4-amino-6,7-di-substituted pteridine derivative can beobtained in step (c) by reacting a2-R₂-substituted-4,5,6-triamino-pyrimidine with a disubstituted glyoxalbearing the groups R₃ and R₄, wherein each of R₃ and R₄ is independentlyselected (i.e. R₃ and R₄ may be identical or different) from the groupconsisting of C₁₋₇ alkyl, C₃₋₁₀ cycloalkyl, aryl or heteroaryl, underneutral or basic conditions. In step (d), acidic or basic hydrolysis ofthe amino group at position 4 of the pteridine ring is performed andresults in the corresponding 4-oxo-pteridine derivative. In step (e),the hydroxyl group of the tautomeric form of the latter is activated fora nucleophilic displacement reaction, e.g. by preparing the4-[(1,2,4)triazolyl]pteridine derivative. In the last step (f), the4-triazolylpteridine derivative is reacted with a nucleophile having thegeneral formula R₁XH, such as for example a primary or secondary amine,C₁₋₇ alkoxy, aryloxy, C₃₋₁₀ cycloalkoxy, heteroaryloxy, mercapto C₁₋₇alkyl, mercaptoaryl, mercapto C₃₋₁₀ cycloalkyl, or mercapto-heteroaryl.

FIG. 5 represents a scheme for the synthesis of symmetrical2,4,6-trisubstituted and 2,4,7-trisubstituted, as well as2,4,6,7-tetrasubstituted pteridine derivatives with various R₁, R₂, R₃and/or R₄ substituents in the 2-, 4-, 6- and/or 7-positions of thepteridine ring. In step (a), a nitroso group is introduced on position 5of the pyrimidine ring of a 2-R₂-substituted 4-oxo-6-aminopyrimidine byusing sodium nitrite under aqueous acidic conditions. Reduction of thenitroso group in step (b) is achieved either catalytically (Pt/H₂) inthe presence of a protic solvent, or chemically using sodium dithioniteor ammonium sulfide in water. Then in a next step (c), condensing theresulting 2-R₂-substituted 4-oxo-5,6-diamino-pyrimidine with anα-ketoaldoxime bearing a radical R₃, wherein R₃ may be inter alia C₁₋₇alkyl, C₃₋₁₀ cycloalkyl, aryl or heteroaryl, in a protic solvent such asmethanol under acidic conditions regioselectively yields a4-oxopteridine bearing a R₂ substituent in position 2 and a R₃substituent in position 6 of the pteridine ring. Alternatively, a2-R₂-substituted 4-oxo-7-R₄-substituted pteridine derivative can beobtained in step (d) by reacting the 2-R₂-substituted4-oxo-5,6-diaminopyrimidine with a monosubstituted glyoxal bearing thegroup R₄, wherein R₄ may be inter alia C₁₋₇ alkyl, C₃₋₁₀ cycloalkyl,aryl or heteroaryl, under neutral or basic conditions. Alternatively, a2-R₂-substituted-4-oxo-6,7-disubstituted pteridine derivative can beobtained in step (e) by reacting the 2-R₂-substituted4-oxo-5,6-diamino-pyrimidine with a disubstituted glyoxal bearing groupsR₃ and R₄, wherein each of R₃ and R₄ is independently selected (i.e. R₃and R₄ may be identical or different) from the group consisting of C₁₋₇alkyl, C₃₋₁₀ cycloalkyl, aryl and heteroaryl, under neutral or basicconditions. Activation of the (tautomeric) hydroxyl substituent inposition 4 of the pteridine ring for a nucleophilic displacementreaction occurs in step (f) by preparing the corresponding4-[(1,2,4)-triazolyl] pteridine derivative, e.g. using POCl₃ or4-chlorophenyl phosphorodichloridate and 1,2,4-triazole in pyridine assolvent. When R₂ is an amino group, protection of R₂ may further benecessary before carrying out this reaction. The amino group can beprotected for instance by an acetyl group, which can be hydrolysed backto the amino group in a next step. Nucleophilic substitution isperformed in step (g) by mixing the triazolyl pteridine derivative witha nucleophile R₁XH, (such as for example such as for example a primaryor secondary amine, C₁₋₇ alkoxy, aryloxy, C₃₋₁₀ cycloalkoxy,heteroaryloxy, mercapto C₁₋₇ alkyl, mercaptoaryl, mercapto C₃₋₁₀cycloalkyl, or mercapto-heteroaryl) at room temperature in a polaraprotic solvent such as 1,4-dioxane.

FIG. 6 represents a scheme for the preparation of symmetrical2,4,6-trisubstituted pteridines and 2,4,7-trisubstituted as well as2,4,6,7-tetrasubstituted pteridine derivatives, i.e. whereinsubstituents in the 2- and 4-positions of the pteridine ring areidentical. In step (a), a nitro group is introduced in position 5 of a6-amino-2,4-dioxopyrimidine under strongly acidic conditions (e.g. HNO₃,H₂SO₄). Then, in step (b), both hydroxyl groups from the tautomeric formare converted to chloro groups by treatment with a chlorinating agentsuch as POCl₃ or SOCl₂. Both chloro groups are then displaced in step(c) with a nucleophile having the general formula R₁XH. The nitro groupof the latter is then reduced in step (d) to an amino group by treatmentwith a reducing agent (e.g. Pt/H₂). Finally, reaction of the latter withan α-ketoaldoxime bearing the group R₃, wherein R₃ may be inter aliaaryl, C₁₋₇ alkyl, C₁₋₁₀ cycloalkyl or heteroaryl, regioselectivelyyields the desired 2,4,6-trisubstituted pteridine derivative in step(e). Alternatively, reaction of the2,4-substituted-5,6-diaminopyrimidine from step (d) with amonosubstituted glyoxal bearing a group R₄ wherein R₄ may be inter aliaC₁₋₇ alkyl, C₃₋₁₀ cycloalkyl, aryl or heteroaryl yields the desired2,4,7-trisubstituted pteridine derivative in step (f). Alternatively,reaction of the 2,4-substituted-5,6-diaminopyrimidine from step (d) witha disubstituted glyoxal bearing groups R₃ and R₄, wherein each of R₃ andR₄ is independently selected (i.e. R₃ and R₄ may be identical ordifferent) from the group consisting of C₁₋₇ alkyl, C₃₋₁₀ cycloalkyl,aryl and heteroaryl, under neutral or basic conditions, yields thedesired 2,4,6,7-tetrasubstituted pteridine derivative in step (g).

FIG. 7 represents a scheme for the preparation of 2,4,6-trisubstitutedpteridine derivatives, wherein the substituent in the 6-position of thepteridine ring is a phenyl group substituted by a nitrogen-containingfunction. In step (a), the chlorine at position 4 of the pyrimidine ringis displaced by an appropriate nucleophile, having the general formulaR₁XH (such as, but not limited to, a primary or secondary amine, C₁₋₇alkoxy, aryloxy, C₃₋₁₀ cycloalkoxy, heteroaryloxy, mercapto C₁₋₇ alkyl,mercaptoaryl, mercapto C₃₋₁₀ cycloalkyl, or mercapto-heteroaryl).Introduction of the nitroso group at position 5 of the pyrimidine ringis achieved by treatment with sodium nitrite under aqueous acidicconditions. Reduction of the nitroso group in step (c) is achievedeither catalytically (Pt/H₂) in the presence of a protic solvent, orchemically using sodium dithionite or ammonium sulfide in water. In step(d), the 2-R₂-4-XR₁-substituted-5,6-triamino-pyrimidine analogue iscondensed with an acetamidophenylglyoxalmonoxime in a protic solventsuch as methanol under acidic conditions to yield regioselectively apteridine analogue bearing an acetamidophenyl group at position 6 of thepteridine ring. Acidic deprotection of the acetyl group leads to theformation of the free amino group. This amino group can be transformedinto amides (by reaction with carboxylic acids or acid chlorides) orinto sulfonamides (by reaction with sulfonyl chlorides) by reaction in apolar aprotic solvent (e.g. pyridine, dimethylformamide ordichloromethane) and optionally further in the presence of a base (e.g.triethylamine).

FIG. 8 represents a scheme for the preparation of 2,4,6-trisubstitutedpteridine derivatives, wherein the substituent in the 6-position of thepteridine ring is a phenyl group substituted by an oxygen-containingfunction. In step (a) a 2-R₂-4-XR₁-substituted-5,6-triaminopyrimidine,which may be obtained for instance after step (c) described in FIG. 7,is condensed with an hydroxyphenyl-glyoxalmonoxime in a protic solventsuch as methanol under acidic conditions to yield regioselectively apteridine analogue bearing an hydroxyphenyl group at position 6 of thepteridine ring. The free hydroxyl group can be alkylated in a polarprotic solvent (e.g. dimethylformamide) using a base (such as, but notlimited to, potassium carbonate, cesium carbonate or sodium hydride) andan appropriate alkylhalide or arylalkylhalide.

Some sub-sets of pteridine derivatives according to the inventiondeserve specific interest. Thus in a particular embodiment the inventionrelates to a group of pteridine derivatives, as well as pharmaceuticalcompositions comprising such pteridine derivatives as active principle,having the above general formula (I) wherein:

-   -   X is NZ,    -   XR₁ is selected from the group consisting of hydroxylamino;        hydrazino (i.e. Z is hydrogen and R₁ is amino); (mono- or di-)        C₁₋₇ alkylamino (i.e. R₁ is C₁₋₇ alkyl); (mono- or di-)        arylamino (i.e. R₁ is aryl, e.g. adamantyl); (mono- or di-)        C₃₋₁₀ cycloalkylamino (i.e. R₁ is C₃₋₁₀ cycloalkyl); (mono- or        di-)hydroxyC₁₋₇ alkylamino (i.e. R₁ is C₁₋₇ alkyl substituted        with hydroxyl); (mono- or di-) C₁₋₄ alkylarylamino; and        saturated or unsaturated heterocyclic groups containing at least        one nitrogen atom and optionally substituted by one or more        substituents independently selected from the group consisting of        C₁₋₄ alkyl, hydroxy C₁₋₄ alkyl, C₁₋₄ alkyloxy, halogen, hydroxy,        hydroxycarbonyl and C₁₋₄ alkyloxycarbonyl, such as but not        limited to piperazinyl, N-alkylpiperazinyl and morpholinyl;    -   R₂ is amino or is selected from the group consisting of        hydroxylamino; hydrazino (i.e. Z is hydrogen and R₁ is amino);        (mono- or di-)arylamino (i.e. R₁ is aryl); (mono- or di-) C₃₋₁₀        cycloalkylamino (i.e. R₁ is C₃₋₁₀ cycloalkyl); (mono- or        di-)hydroxyC₁₋₇ alkylamino (i.e. R₁ is C₁₋₇ alkyl substituted        with hydroxyl; (mono- or di-) C₁₋₄ alkylarylamino; and saturated        or unsaturated heterocyclic groups containing at least one        nitrogen atom and optionally substituted by one or more        substituents independently selected from the group consisting of        C₁₋₄ alkyl, hydroxy C₁₋₄ alkyl, C₁₋₄ alkyloxy, halogen, hydroxy,        hydroxycarbonyl and C₁₋₄ alkyloxycarbonyl, such as but not        limited to piperazinyl, N-alkylpiperazinyl and morpholinyl;    -   R₃ is selected from the group consisting of unsubstituted,        monosubstituted and disubstituted aryl groups (wherein the        substituent(s) may indepen-dently be halogen, C₁₋₄ alkoxy or        C₁₋₄ alkyl); 3,4,5-trimethoxyphenyl;        3,4-formylidene-3,4-dihydroxyphenyl; aryl groups bonded to the        pteridine ring via a saturated or unsaturated aliphatic spacer        which may be halogenated or hydroxylated; and aliphatic        substituents which may contain ether function, alcohol function,        or substituted or unsubstituted amino functions or C₁₋₄        alkyloxy; and    -   R₄ is selected from the group consisting of hydrogen, alkyl and        alkoxy;        or a pharmaceutically acceptable addition salt or a        stereochemical isomeric form thereof.

In another particular embodiment, the invention relates to a group ofpteridine derivatives, as well as pharmaceutical compositions comprisingsuch pteridine derivatives as active principle, having the above generalformula (I) wherein:

-   -   X is oxygen or sulfur;    -   R₁ is C₁₋₇ alkyl;    -   R₂ is amino or is selected from the group consisting of        hydroxylamino; hydrazino (i.e. Z is hydrogen and R₁ is amino);        (mono- or di-)arylamino (i.e. R₁ is aryl); (mono- or di-) C₃₋₁₀        cycloalkylamino (i.e. R₁ is C₃₋₁₀ cycloalkyl); (mono- or        di-)hydroxyC₁₋₇ alkylamino (i.e. R₁ is C₁₋₇ alkyl substituted        with hydroxyl); (mono- or di-) C₁₋₄ alkylarylamino; and        saturated or unsaturated heterocyclic groups containing at least        one nitrogen atom and optionally substituted by one or more        substituents independently selected from the group consisting of        C₁₋₄ alkyl, hydroxy C₁₋₄ alkyl, C₁₋₄ alkyloxy, halogen, hydroxy,        hydroxycarbonyl and C₁₋₄ alkyloxycarbonyl, such as but not        limited to piperazinyl, N-alkylpiperazinyl and morpholinyl;    -   R₃ is selected from the group consisting of unsubstituted,        monosubstituted and disubstituted aryl groups (wherein the        substituent(s) may indepen-dently be halogen, C₁₋₄ alkoxy or        C₁₋₄ alkyl); aryl groups bonded to the pteridine ring via a        saturated or unsaturated aliphatic spacer which may be        halogenated or hydroxylated; and aliphatic substituents which        may contain ether function, alcohol function, or substituted or        unsubstituted amino functions or C₁₋₄ alkyloxy; and    -   R₄ is selected from the group consisting of hydrogen, alkyl and        alkoxy;        or a pharmaceutically acceptable addition salt or a        stereochemical isomeric form thereof.

In another particular embodiment, the invention relates to a group ofpteridine derivatives having the above general formula (I) wherein Xrepresents an oxygen atom or a group with the formula S(O)_(m), whereinm is an integer from 0 to 2, or a group with the formula NZ and wherein:

-   -   R₁ is a group selected from the group consisting of C₁₋₄ alkyl,        C₂₋₇ alkenyl, C₂₋₇ alkynyl, C₃₋₁₀ cycloalkyl, C₃₋₁₀        cycloalkenyl, aryl, alkylaryl, arylalkyl, heterocyclic,        heterocyclic-substituted alkyl and alkyl-substituted        heterocyclic, each of said groups being optionally substituted        with one or more substituents selected from the group consisting        of halogen, C₁₋₄ alkyl, C₁₋₄ alkoxy, C₂₋₇ alkenyl, C₂₋₇ alkynyl,        halo C₁₋₄ alkyl, C₃₋₁₀ cycloalkoxy, aryloxy, arylalkyloxy,        oxyheterocyclic, heterocyclic-substituted alkyloxy, thio C₁₋₇        alkyl, thio C₃₋₁₀ cycloalkyl, thioaryl, thioheterocyclic,        arylalkylthio, heterocyclic-substituted alkylthio, formyl,        hydroxyl, sulfhydryl, nitro, hydroxylamino, mercaptoamino,        cyano, carboxylic acid or esters or thioesters or amides or        thioamides or halides or anhydrides thereof, thiocarboxylic acid        or esters or thioesters or amides or thioamides or halides or        anhydrides thereof, carbamoyl, thiocarbamoyl, ureido,        thio-ureido, amino, cycloalkylamino, alkenylamino,        cycloalkenylamino, alkynylamino, arylamino, arylalkylamino,        hydroxylalkylamino, mercaptoalkyl-amino, heterocyclic amino,        hydrazino, alkylhydrazino and phenyl-hydrazino; or R₁ is a        carboxyalkyl, carboxyaryl, thiocarboxyaryl or thiocarboxyalkyl        group;    -   Z is a group independently defined as R₁ or Z is hydrogen or the        group NZ together with R₁ is either hydroxylamino or an        optionally substituted heterocyclic group containing at least        one nitrogen atom;    -   R₂ is selected from the group consisting of amino; acylamino;        thioacylamino; carbamoyl; thiocarbamoyl, ureido; thio-ureido,        sulfonamido; hydroxylamino; alkoxyamino; thioalkylamino;        mercaptoamino, hydrazino; alkylhydrazino; phenylhydrazino;        optionally substituted heterocyclic radicals; C₃₋₇ alkylamino;        arylamino; arylalkyl-amino; cycloalkylamino; alkenylamino;        cycloalkenylamino; heterocyclic amino; hydroxyalkylamino;        mercaptoalkylamino; C₁₋₇ alkoxy; C₃₋₁₀ cycloalkoxy; thio C₁₋₇        alkyl; arylsulfoxide; arylsulfone; heterocyclic sulfoxide;        heterocyclic sulfone; thio C₃₋₁₀ cycloalkyl; aryloxy; arylthio;        arylalkyloxy; arylalkylthio; oxyheterocyclic and        thioheterocyclic radicals;    -   R₄ is an atom or a group selected from the group consisting of        hydrogen; halogen; C₁₋₇ alkyl; C₂₋₇ alkenyl; C₂₋₇ alkynyl; halo        C₁₋₇ alkyl; carboxy C₁₋₇ alkyl; carboxyaryl; C₁₋₇ alkoxy; C₃₋₁₀        cycloalkoxy; aryloxy; arylalkyloxy; oxyheterocyclic;        heterocyclic-substituted alkyloxy; thio C₁₋₇ alkyl; thio C₃₋₁₀        cycloalkyl; thioaryl; thioheterocyclic; arylalkylthio;        heterocyclic substituted alkylthio; hydroxylamino;        mercapto-amino; acylamino; thio-acylamino; alkoxyamino;        thioalkylamino; acetal; thio-acetal; carboxylic acid; carboxylic        acid esters, thioesters, halides, anhydrides, amides and        thioamides; thiocarboxylic acid; thiocarboxylic acid esters,        thioesters, halides, anhydrides, amides and thioamides;        hydroxyl; sulfhydryl; nitro; cyano; carbamoyl; thiocarbamoyl,        ureido; thio-ureido; alkylamino; cycloalkylamino; alkenylamino;        cycloalkenylamino; alkynylamino; arylamino; arylalkylamino;        hydroxyalkylamino; mercaptoalkylamino; heterocyclic amino;        heterocyclic-substituted alkylamino; oximino; alkyloximino;        hydrazino; alkylhydrazino; phenylhydrazino; cysteinyl acid,        esters, thioesters, halides, anhydrides, amides and thioamides        thereof; aryl being optionally substituted with one or more        substituents selected from the group consisting of halogen, C₁₋₇        alkyl, C₁₋₇ alkoxy, C₂₋₇ alkenyl, C₂₋₇ alkynyl, halo C₁₋₇ alkyl,        nitro, hydroxyl, sulfhydryl, amino, C₃₋₁₀ cycloalkoxy, aryloxy,        arylalkyloxy, oxyheterocyclic, heterocyclic-substituted        alkyloxy, thio C₁₋₇ alkyl, thio C₃₋₁₀ cycloalkyl, thioaryl,        thioheterocyclic, arylalkylthio, heterocyclic-substituted        alkylthio, formyl, carbamoyl, thiocarbamoyl, ureido,        thio-ureido, sulfonamido, hydroxylamino, alkoxyamino,        mercaptoamino, thioalkylamino, acylamino, thioacyl-amino, cyano,        carboxylic acid or esters or thioesters or halides or anhydrides        or amides thereof, thiocarboxylic acid or esters or thioesters        or halides or anhydrides or amides thereof, alkylamino,        cycloalkylamino, alkenylamino, cycloalkenylamino, alkynylamino,        arylamino, arylalkylamino, hydroxyalkylamino,        mercaptoalkylamino, heterocyclic amino, hydrazino,        alkylhydrazino and phenylhydrazino; optionally substituted        heterocyclic radicals; aromatic or heterocyclic substituents        substituted with an aliphatic spacer between the pteridine ring        and the aromatic or heterocyclic substituent, whereby said        aliphatic spacer is a branched or straight, saturated or        unsaturated aliphatic chain of 1 to 4 carbon atoms which may        contain one or more functions, atoms or radicals selected from        the group consisting of carbonyl (oxo), thiocarbonyl, alcohol        (hydroxyl), thiol, ether, thio-ether, acetal, thio-acetal,        amino, imino, oximino, alkyloximino, amino-acid, cyano,        acylamino, thioacylamino, carbamoyl, thiocarbamoyl, ureido,        thio-ureido, carboxylic acid or ester or thioester or halide or        anhydride or amide, thiocarboxylic acid or ester or thioester or        halide or anhydride or amide, nitro, thio C₁₋₇ alkyl, thio C₃₋₁₀        cycloalkyl, hydroxylamino, mercaptoamino, alkylamino,        cycloalkylamino, alkenylamino, cycloalkenyl-amino, alkynylamino,        arylamino, arylalkylamino, hydroxyalkylamino,        mercaptoalkylamino, heterocyclic amino, hydrazino,        alkylhydrazino, phenylhydrazino, sulfonyl, sulfinyl, sulfonamido        and halogen; branched or straight, saturated or unsaturated        aliphatic chains of 1 to 7 carbon atoms optionally containing        one or more functions selected from the group consisting of        carbonyl (oxo), thiocarbonyl, alcohol (hydroxyl), thiol, ether,        thio-ether, acetal, thio-acetal, amino, imino, oximino,        alkyloximino, aminoacid, cyano, acylamino; thioacylamino;        carbamoyl, thiocarbamoyl, ureido, thio-ureido, carboxylic acid        ester or halide or anhydride or amide, thiocarboxylic acid or        ester or thioester or halide or anhydride or amide, nitro, thio        C₁₋₇ alkyl, thio C₃₋₁₀ cycloalkyl, hydroxylamino, mercaptoamino,        alkylamino, cycloalkylamino, alkenylamino, cycloalkenylamino,        alkynyl-amino, arylamino, arylalkylamino, hydroxyalkylamino,        mercaptoalkylamino, heterocyclic amino, hydrazino,        alkylhydrazino, phenylhydrazino, sulfonyl, sulfinyl, sulfonamido        and halogen; and    -   R₃ is selected from chloro and thiomethyl;        and/or a pharmaceutically acceptable addition salt thereof        and/or a stereoisomer thereof and/or a mono- or a di-N-oxide        thereof and/or a solvate and/or a dihydro- or        tetrahydropteridine derivative thereof.

When applicable, and depending upon the specific substituents beingpresent, not only the novel pteridine having the general formula (I) butalso some pteridine derivatives previously known in the art without anyindication of biological activity, i.e. all of the pteridines having thegeneral formula (II) according to this invention, may be in the form ofa pharmaceutically acceptable salt. The latter include anytherapeutically active non-toxic addition salt which compounds havingthe general formula (II) are able to form with a salt-forming agent.Such addition salts may conveniently be obtained by treating thepteridine derivatives of the invention with an appropriate salt-formingacid or base. For instance, pteridine derivatives having basicproperties may be converted into the corresponding therapeuticallyactive, non-toxic acid addition salt form by treating the free base formwith a suitable amount of an appropiate acid following conventionalprocedures. Examples of such appropriate salt-forming acids include, forinstance, inorganic adds resulting in forming salts such as but notlimited to hydrohalides (e.g. hydrochloride and hydrobromide), sulfate,nitrate, phosphate, diphosphate, carbonate, bicarbonate, and the like;and organic monocarboxylic or dicarboxylic acids resulting in formingsalts such as, for example, acetate, propanoate, hydroxyacetate,2-hydroxypropanoate, 2-oxopropanoate, lactate, pyruvate, oxalate,malonate, succinate, maleate, fumarate, malate, tantrate, citrate,methanesulfonate, ethanesulfonate, benzoate, 2-hydroxybenzoate,4-amino-2-hydroxybenzoate, benzene-sulfonate, p-toluenesulfonate,salicylate, p-aminosalicylate, pamoate, bitartrate, camphorsulfonate,edetate, 1,2-ethanedisulfonate, fumarate, glucoheptonate, gluconate,glutamate, hexylresorcinate, hydroxynaphtoate, hydroxyethanesulfonate,mandelate, methylsulfate, pantothenate, stearate, as well as saltsderived from ethanedioic, propanedioic, butanedioic, (Z)-2-butenedioic,(E)-2-butenedioic, 2-hydroxybutanedioic, 2,3-dihydroxy-butanedioic,2-hydroxy-1,2,3-propanetricarboxylic and cyclohexanesulfamic acids andthe like.

Pteridine derivatives having acidic properties may be converted in asimilar manner into the corresponding therapeutically active, non-toxicbase addition salt form. Examples of appropriate salt-forming basesinclude, for instance, inorganic bases like metallic hydroxides such asbut not limited to those of alkali and alkaline-earth metals likecalcium, lithium, magnesium, potassium and sodium, or zinc, resulting inthe corresponding metal salt; organic bases such as but not limited toammonia, alkylamines, benzathine, hydrabamine, arginine, lysine,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, N-methylglucamine, procaine and the like.

Reaction conditions for treating the pteridine derivatives (II) of thisinvention with an appropriate salt-forming acid or base are similar tostandard conditions involving the same acid or base but differentorganic compounds with basic or acidic properties, respectively.Preferably, in view of its use in a pharmaceutical composition or in themanufacture of medicament for treating specific diseases, thepharmaceutically acceptable salt will be designed, i.e. the salt-formingacid or base will be selected so as to impart greater water-solubility,lower toxicity, greater stability and/or slower dissolution rate to thepteridine derivative of this invention.

The present invention further provides the use of a pteridine derivativerepresented by the general formula (II), or a pharmaceuticallyacceptable salt or a solvate thereof, as a biologically-activeingredient, i.e. an active principle, especially as a medicine or adiagnostic agent or for the manufacture of a medicament or a diagnostickit. In particular the said medicament may be for the prevention ortreatment of a pathologic condition selected from the group consistingof:

-   -   immune disorders, in particular organ and cells transplant        rejections, and autoimmune disorders,    -   cardiovascular disorders,    -   allergic conditions,    -   disorders of the central nervous system, and    -   cell proliferative disorders.

The pathologic conditions and disorders concerned by the said use, andthe corresponding methods of prevention or treatment, are detailedhereinbelow. Any of the uses mentioned with respect to the presentinvention may be restricted to a non-medical use (e.g. in a cosmeticcomposition), a non-therapeutic use, a non-diagnostic use, a non-humanuse (e.g. in a veterinary composition), or exclusively an in-vitro use,or a use with cells remote from an animal.

The invention further relates to a pharmaceutical compositioncomprising:

-   (a) one or more pteridine represented by the general formula (II),    and-   (b) one or more pharmaceutically acceptable carriers.

In a third embodiment, this invention provides combinations, preferablysynergistic combinations, of one or more pteridine represented by thegeneral formula (II) with one or more biologically-active drugs beingpreferably selected from the group consisting of immunosuppressantand/or immunomodulator drugs, antineoplastic drugs, antihistamines,inhibitors of allergy-causative agents (anti-allergic drugs) andantiviral agents. As is conventional in the art, the evaluation of asynergistic effect in a drug combination may be made by analyzing thequantification of the interactions between individual drugs, using themedian effect principle described by Chou et al. in Adv. Enzyme Reg.(1984) 22:27. Briefly, this principle states that interactions(synergism, additivity, antagonism) between two drugs can be quantifiedusing the combination index (hereinafter referred as Cl) defined by thefollowing equation:

${CI}_{x} = {\frac{{ED}_{x}^{1c}}{{ED}_{x}^{1a}} + \frac{{ED}_{x}^{2c}}{{ED}_{x}^{2a}}}$wherein ED_(x) is the dose of the first or respectively second drug usedalone (1a, 2a), or in combination with the second or respectively firstdrug (1c, 2c), which is needed to produce a given effect. The said firstand second drug have synergistic or additive or antagonistic effectsdepending upon Cl<1, Cl=1, or Cl>1, respectively. As will be explainedin more detail herein-below, this principle may be applied to a numberof desirable effects such as, but not limited to, an activity againsttransplant rejection, an activity against immunosuppression orimmunomodulation, an activity against allergy or an activity againstcell proliferation.

For instance the present invention relates to a pharmaceuticalcomposition or combined preparation having synergistic effects againstimmuno-suppression or immunomodulation and containing:

-   (a) one or more immunosuppressant and/or immunomodulator drugs, and-   (b) at least one pteridine derivative represented by the general    formula (II), and-   (c) optionally one or more pharmaceutical excipients or    pharmaceutically acceptable carriers, for simultaneous, separate or    sequential use in the treatment or prevention of autoimmune    disorders and/or in transplant-rejections.

Suitable immunosuppressant drugs for inclusion in the synergisticcompositions or combined preparations of this invention belong to a wellknown therapeutic class. They are preferably selected from the groupconsisting of cyclosporin A, substituted xanthines (e.g. methylxanthinessuch as pentoxyfylline), daltroban, sirolimus, tacrolimus, rapamycin(and derivatives thereof such as defined below), leflunomide (or itsmain active metabolite A771726, or analogs thereof calledmalononitrilamides), mycophenolic acid and salts thereof (including thesodium salt marketed under the trade name Mofetil®), adrenocorticalsteroids, azathioprine, brequinar, gusperimus, 6-mercaptopurine,mizoribine, chloroquine, hydroxychloroquine and monoclonal antibodieswith immunosuppressive properties (e.g. etanercept, infliximab orkineret). Adrenocortical steroids within the meaning of this inventionmainly include glucocorticoids such as but not limited to ciprocinonide,desoxycorticisterone, fludrocortisone, flumoxonide, hydrocortisone,naflocort, procinonide, timobesone, tipredane, dexamethasone,methylprednisolone, methotrexate, prednisone, prednisolone,triamcinolone and pharmaceutically acceptable salts thereof. Rapamycinderivatives as referred herein include O-alkylated derivatives,particularly 9-deoxorapamycins, 26-dihydrorapamycins, 40-O-substitutedrapamycins and 28,40-O,O-disubstituted rapamycins (as disclosed in U.S.Pat. No. 5,665,772) such as 40-O-(2-hydroxy)ethyl rapamycin—also knownas SDZ-RAD-, pegylated rapamycin (as disclosed in U.S. Pat. No.5,780,462), ethers of 7-desmethylrapamycin (as disclosed in U.S. Pat.No. 6,440,991) and polyethylene glycol esters of SDZ-RAD (as disclosedin U.S. Pat. No. 6,331,547).

Suitable immunomodulator drugs for inclusion into the synergisticimmunomodulating pharmaceutical compositions or combined preparations ofthis invention are preferably selected from the group consisting ofacemannan, amiprilose, bucillamine, dimepranol, ditiocarb sodium,imiquimod, Inosine Pranobex, interferon-β, interferon-γ, lentinan,levamisole, lisophylline, pidotimod, romurtide, platonin, procodazole,propagermanium, thymomodulin, thymopentin and ubenimex.

Synergistic activity of the pharmaceutical compositions or combinedpreparations of this invention against immunosuppression orimmuno-modulation may be readily determined by means of one or morelymphocyte activation tests. Usually activation is measured vialymphocyte proliferation. Inhibition of proliferation thus always meansimmunosuppression under the experimental conditions applied. There existdifferent stimuli for lymphocyte activation, in particular:

-   a) co-culture of lymphocytes of different species (mixed lymphocyte    reaction, hereinafter referred as MLR) in a so-called mixed    lymphocyte culture test: lymphocytes expressing different minor and    major antigens of the HLA-DR type (=alloantigens) activate each    other non-specifically;-   b) a CD3 assay wherein there is an activation of the T-lymphocytes    via an exogenously added antibody (OKT3). This antibody reacts    against a CD3 molecule located on the lymphocyte membrane which has    a co-stimulatory function. Interaction between OKT3 and CD3 results    in T-cell activation which proceeds via the    Ca²⁺/calmodulin/calcineurin system and can be inhibited e.g. by    cyclosporin A (hereinafter referred as CyA);-   c) a CD28 assay wherein specific activation of the T-lymphocyte    proceeds via an exogenously added antibody against a CD28 molecule    which is also located on the lymphocyte membrane and delivers strong    co-stimulatory signals. This activation is Ca²⁺-independent and thus    cannot be inhibited by CyA.

Determination of the immunosuppressing or immunomodulating activity ofthe pteridine derivatives of this invention, as well as synergisticcombinations comprising them, is preferably based on the determinationof one or more, preferably at least three lymphocyte activation in vitrotests, more preferably including at least one of the MLR test, CD3 assayand CD28 assay referred above. Preferably the lymphocyte activation invitro tests used include at least two assays for two different clustersof differentiation preferably belonging to the same general type of suchclusters and more preferably belonging to type I transmembrane proteins.Optionally the determination of the immuno-suppressing orimmunomodulating activity may be performed on the basis of otherlymphocyte activation in vitro tests, for instance by performing a TNF-αassay or an IL-1 assay or an IL-6 assay or an IL-10 assay or an IL-12assay or an assay for a cluster of differentiation belonging to afurther general type of such clusters and more preferably belonging totype II transmembrane proteins such as, but not limited to, CD69, CD 71or CD134.

The synergistic effect may be evaluated by the median effect analysismethod described herein-before. Such tests may for instance, accordingto standard practice in the art, involve the use of equiment, such asflow cytometer, being able to separate and sort a number of cellsubcategories at the end of the analysis, before these purified batchescan be analysed further.

Synergistic activity of the pharmaceutical compositions of thisinvention in the prevention or treatment of transplant rejection may bereadily determined by means of one or more leukocyte activation testsperformed in a Whole Blood Assay (hereinafter referred as WBA) describedfor instance by Lin et al. in Transplantation (1997) 63:1734-1738. WBAused herein is a lymphoproliferation assay performed in vitro usinglymphocytes present in the whole blood, taken from animals that werepreviously given the pteridine derivative, and optionally the otherimmunosuppressant drug, in vivo (more details are given in example 118).Hence this assay reflects the in vivo effect of substances as assessedby an in vitro read-out assay. The synergistic effect may be evaluatedby the median effect analysis method described herein-before. Variousorgan transplantation models in animals are also available in vivo,which are strongly influenced by different immunogenicities, dependingon the donor and recipient species used and depending on the nature ofthe transplanted organ. The survival time of transplanted organs canthus be used to measure the suppression of the immune response.

The pharmaceutical composition or combined preparation with synergisticactivity against immunosuppression or immunomodulation according to thisinvention may contain the pteridine derivative of formula (II) over abroad content range depending on the contemplated use and the expectedeffect of the preparation. Generally, the pteridine derivative contentof the combined preparation is within the range of 0.1 to 99.9% byweight, preferably from 1 to 99% by weight, more preferably from 5 to95% by weight.

The invention further relates to a composition or combined preparationhaving synergistic effects against cell proliferation and containing:

-   (a) one or more antineoplastic drugs, and-   (b) at least one pteridine deivative represented by the general    formula (II), and-   (c) optionally one or more pharmaceutical excipients or    pharmaceutically acceptable carriers, for simultaneous, separate or    sequential use in the treatment or prevention of cell proliferative    disorders.

Suitable antineoplastic drugs for inclusion into the synergisticantiproliferative pharmaceutical compositions or combined preparationsof this invention are preferably selected from the group consisting ofalkaloids, alkylating agents (including but not limited to alkylsulfonates, aziridines, ethylenimines, methylmelamines, nitrogenmustards and nitrosoureas), antibiotics, antimetabolites (including butnot limited to folic acid analogs, purine analogs and pyrimidineanalogs), enzymes, interferon and platinum complexes. More specificexamples include acivicin; aclarubicin; acodazole; acronine; adozelesin;aldesleukin; altretamine; ambomycin; ametantrone; aminoglutethimide;amsacrine; anastrozole; anthramycin; asparaginase; asperlin;azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide;bisantrene; bisnafide; bizelesin; bleomycin; brequinar; bropirimine;busulfan; cactinomycin; calusterone; caracemide; carbetimer;carboplatin; carmustine; carubicin; carzelesin; cedefingol;chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol;cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin;decitabine; dexormaplatin; dezaguanine; diaziquone; docetaxel;doxorubicin; droloxifene; dromostanolone; duazomycin; edatrexate;eflomithine; elsamitrucin; enloplatin; enpromate; epipropidine;epirubicin; erbulozole; esorubicin; estramustine; etanidazole;ethiodized oil I 131; etoposide; etoprine; fadrozole; fazarabine;fenretinide; floxuridine; fludarabine; fluorouracil; flurocitabine;fosquidone; fostriecin; gemcitabine; Gold 198; hydroxyurea; idarubicin;ifosfamide; ilmofosine; interferon α-2a; interferon α-2b; interferonα-n1; interferon α-n3; interferon β-1a; interferon γ-1b; iproplatin;irinotecan; lanreotide; letrozole; leuprolide; liarozole; lometrexol;lomustine; losoxantrone; masoprocol; maytansine; mechlorethamine;megestrol; melengestrol; melphalan; menogaril; mercaptopurine;methotrexate; metoprine; meturedepa; mitindomide; mitocarcin;mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane;mitoxantrone; mycophenolic add; nocodazole; nogala-mycin; ormaplatin;oxisuran; paclitaxel; pegaspargase; pellomycin; pentamustine;peplomycin; perfosfamide; pipobroman; piposulfan; piroxantrone;plicamycin; plomestane; porfimer, porfiromycin; prednimustine;procarbazine; puromycin; pyrazofurin; riboprine; rogletimide; safingol;semustine; simtrazene; sparfosate; sparsomycin; spirogermanium;spiromustine; spiroplatin; streptonigrin; streptozocin; strontium 89chloride; sulofenur; talisomycin; taxane; taxoid; tecogalan; tegafur;teloxantrone; temoporfin; teniposide; teroxirone; testolactone;thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; topotecan;toremifene; trestolone; triciribine; trimetrexate; triptorelin;tubulozole; uracil mustard; uredepa; vapreotide; verteporFin;vinblastine; vincristine; vindesine; vinepidine; vinglycinate;vinleurosine; vinorelbine; vinrosidine; vinzolidine; vorozole;zeniplatin; zinostatin; zorubidn; and their pharmaceutically acceptablesalts.

Other suitable anti-neoplastic compounds include 20-epi-1,25dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin;acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists;altretamine; ambamustine; amidox; amifostine; aminolevulinic acid;amrubicin; amsacrine; anagrelide; anastrozole; andrographolide;angiogenesis inhibitors; antagonist D; antagonist G; antarelix;anti-dorsalizing morphogenetic protein-1; anti-androgens such as, butnot limited to, benorterone, cioteronel, cyproterone, delmadinone,oxendolone, topterone, zanoterone and their pharmaceutically acceptablesalts; anti-estrogens such as, but not limited to, clometherone;delmadinone; nafoxidine; nitromifene; raloxifene; tamoxifen; toremifene;trioxifene and their phamnaceutically acceptable salts; antineoplaston;antisense oligonucleotides; aphidicolin glycinate; apoptosis genemodulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA;arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin;azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol;batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine;β-lactam derivatives; β-alethine; betaclamycin B; betulinic acid; bFGFinhibitor, bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;bistratene A; bizelesin; breflate; bropirimine; budotitane; buthioninesulfoximine; calcipotriol; calphostin C; camptothecin derivatives;canarypox IL-2; capecitabine; carboxamide-amino-triazole;carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor,carzelesin; casein kinase inhibitors; castanospermine; cecropin B;cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost;cis-porphyrin; clomifene and analogues thereof; clotrimazole; collismydnA and B; combretastatin and analogues thereof; conagenin; crambescidin816; cryptophycin and derivatives thereof; curacin A;cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine; cytolyticfactor; cytostatin; dacliximab; dehydrodidemnin B; deslorelin;dexifosfamide; dexrazoxane; dexverapamil; didemnin B; didox;diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol; dioxamycin;diphenyl spiromustine; docosanol; dolasetron; doxifluridine;droloxifene; dronabinol; duocamnycin SA; ebselen; ecomustine;edelfosine; edrecolomab; elemene; emitefur; epristeride; estrogenagonists and antagonists; exemestane; filgrastim; finasteride;flavopiridol; flezelastine; fluasterone; fluorodaunorunicin; forfenimex;fonmestane; fotemustine; gadolinium texaphyrin; gallium nitrate;galocitabine; ganirelix; gelatinase inhibitors; glutathione inhibitors;hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronicacid; idoxifene; idramantone; ilomastat; imidazoacridones; imiquimod;immunostimulant peptides; insulin-like growth factor-1 receptorinhibitor; interferon agonists; iobenguane; iododoxorubicin; ipomeanol;irinotecan; iroplact; irsogladine; isobengazole; isohomohalicondrin B;itasetron; jasplakinolide; kahalalide F; lamellarin-N; leinamycin;lenograstim; lentinan; leptolstatin; leukemia inhibiting factor;leuprorelin; levamisole; liarozole; lissoclinamide; lobaplatin;lombricine; lonidamine; lovastatin; loxoribine; lurtotecan; lutetiumtexaphyrin; lysofylline; mannostatin A; marimastat; masoprocol; maspin;matrilysin inhibitors; matrix metalloproteinase inhibitors; merbarone;meterelin; methioninase; metoclopramide; MIF inhibitors; mifepristone;miltefosine; mirimostim; mitoguazone; mitolactol; mitonafide; mitotoxinfibroblast growth factor-saporin; mofarotene; molgramostim; humanchorionic gonadotrophin monoclonal antibody; mopidamol; mycaperoxide B;myriaporone; Nacetyldinaline; N-substituted benzamides; nafarelin;nagrestip; naloxone; pentazocine; napavin; naphterpin; nartograstim;nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase;nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant;nitrullyn; octreotide; okicenone; onapristone; ondansetron; ondansetron;oracin; osaterone; oxaliplatin; oxaunomycin; palauamine;palmitoylrhizoxin; pamidronic, add; panaxytriol; panomifene; parabactin;pazelliptine; peldesine; pentosan; pentostatin; pentrozole; perflubron;perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors;picibanil; pilocarpine; pirarubicin; piritrexim; placetin A and B;plasminogen activator inhibitor; propyl bis-acridone; prostaglandin J2;proteasome inhibitors; protein kinase C inhibitors; protein tyrosinephosphatase inhibitors; purine nucleoside phosphorylase inhibitors;purpurins; pyrazoloacridine; raltitrexed; ramosetron; ras farnesylprotein transferase inhibitors; ras inhibitors; ras-GAP inhibitors;retelliptine; rhenium 186 etidronate; rhizoxin; retinamide; rohitukine;romurtide; roquinimex; rubiginone B1; ruboxyl; saintopin; sarcophytol A;sargramostim; sizofuran; sobuzoxane; sodium borocaptate; sodiumphenylacetate; solverol; somatomedin binding protein; sonemnin;sparfosic acid; spicamycin D; splenopentin; spongistatin 1; squalamine;stem-cell division inhibitors; stipiamide; stromelysin inhibitors;sulfinosine; suradista; suramin; swainsonine; tallimustine; tamoxifen;tauromustine; tazarotene; tecogalan; tellurapyrylium; telomeraseinhibitors; temozolomide; tetrachlorodecaoxide; tetrazomine;thaliblastine; thiocoraline; thrombopoietin; thymalfasin; thymopoietinreceptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyletiopurpurin; titanocene; topsentin; tretinoin; triacetyluridine;tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins;ubenimex; urogenital sinus-derived growth inhibitory factor; urokinasereceptor antagonists; variolin B; velaresol; veramine; verdins;verteporfin; vinxaltine; vitaxin; zanoterone; zilascorb; and theirpharmaceutically acceptable salts.

Synergistic activity of the pharmaceutical compositions or combinedpreparations of this invention against cell proliferation may be readilydetermined by means of one or more tests such as, but not limited to,the measurement of the radioactivity resulting from the incorporation of³H-thymidine in culture of tumor cell lines. For instance, differenttumor cell lines are selected in order to evaluate the anti-tumoreffects of the test compounds, such as but not limited to:

-   -   RPMI1788: human Peripheral Blood Leucocytes (PBL) Caucasian        tumor line,    -   Jurkat: human acute T cell leukemia,    -   EL4: C57BI/6 mouse lymphoma, or    -   THP-1: human monocyte tumor line.        Depending on the selected tumor cell line, different culture        media may be used, such as for example:    -   for RPMI1788 and THP-1: RPMI-1640+10% FCS+1% NEAA+1% sodium        pyruvate+5×10⁻⁵ mercapto-ethanol+antibiotics (G-418 0.45 μg/ml).    -   for Jurkat and EL4: RPMI-1640+10% FCS+antibiotics (G-418 0.45        μg/ml).

In a specific embodiment of the synergy determination test, the tumorcell lines are harvested and a suspension of 0.27×10⁶ cells/ml in wholemedium is prepared. The suspensions (150 μl) are added to a microtiterplate in triplicate. Either complete medium (controls) or the testcompounds at the test concentrations (50 μl) are added to the cellsuspension in the microtiter plate. The cells are incubated at 37° C.under 5% CO₂ for about 16 hours. ³H-thymidine is added, and the cellsincubated for another 8 hours. The cells are harvested and radioactivityis measured in counts per minute (CPM) in a β-counter. The ³H-thymidinecell content, and thus the measured radioactivity, is proportional tothe proliferation of the cell lines. The synergistic effect is evaluatedby the median effect analysis method as disclosed herein-before.

The pharmaceutical composition or combined preparation with synergisticactivity against cell proliferation according to this invention maycontain the pteridine compound of formula (II) over a broad contentrange depending on the contemplated use and the expected effect of thepreparation. Generally, the pteridine content of the combinedpreparation is within the range of 0.1 to 99.9% by weight, preferablyfrom 1 to 99% by weight, more preferably from 5 to 95% by weight.

The invention further relates to a pharmaceutical composition orcombined preparation having synergistic effects against a viralinfection and containing:

-   (a) one or more anti-viral agents, and-   (b) at least one pteridine derivative represented by the general    formula (II), and-   (c) optionally one or more pharmaceutical excipients or    pharmaceutically acceptable carriers, for simultaneous, separate or    sequential use in the treatment or prevention of a viral infection.

Suitable anti-viral agents for inclusion into the synergistic antiviralcompositions or combined preparations of this invention include, forinstance, retroviral enzyme Inhibitors belonging to categories wellknown in the art, such as HIV-1 IN inhibitors, nucleoside reversetranscriptase inhibitors (e.g. zidovudine, lamivudine, didanosine,stavudine, zalcitabine and the like), non-nucleoside reversetranscriptase inhibitors (e.g. nevirapine, delavirdine and the like),other reverse transcriptase inhibitors (e.g. foscamet sodium and thelike), and HIV-1 protease inhibitors (e.g. saquinavir, ritonavir,indinavir, nelfinavir and the like). Other suitable antiviral agentsinclude for instance acemannan, acyclovir, adefovir, alovudine,alvircept, amantadine, aranotin, arildone, atevirdine, pyridine,cidofovir, cipamfylline, cytarabine, desciclovir, disoxaril, edoxudine,enviradene, enviroxime, famdclovir, famotine, fiacitabine, fialuridine,floxuridine, fosarilate, fosfonet, ganciclovir, idoxuridine, kethoxal,lobucavir, memotine, methisazone, penciclovir, pirodavir, somantadine,sorivudine, tilorone, trifluridine, valaciclovir, vidarabine, viroxime,zinviroxime, moroxydine, podophyllotoxin, ribavirine, rimantadine,stallimycine, statolon, tromantadine and xenazoic acid, and theirpharmaceutically acceptable salts.

Especially relevant to this aspect of the invention is the inhibition ofthe replication of viruses selected from the group consisting ofpicorna-, toga-, bunya, orthomyxo-, paramyxo-, rhabdo-, retro-, arena-,hepatitis B-, hepatitis C-, hepatitis D, adeno-, vaccinia-, papilloma-,herpes-, corona-, varicella- and zoster-virus, in particular humanimmunodeficiency virus (HIV). Synergistic activity of the pharmaceuticalcompositions or combined preparations of this invention against viralinfection may be readily determined by means of one or more tests suchas, but not limited to, the isobologram method, as previously describedby Elion et al. in J. Biol. Chem. (1954) 208:477-488 and by Baba et al.in Antimicrob. Agents Chemother. (1984) 25:515-517, using EC₅₀ forcalculating the fractional inhibitory concentration (hereinafterreferred as FIC). When the minimum FIC index corresponding to the FIC ofcombined compounds (e.g., FIC_(x)+FIC_(y)) is equal to 1.0, thecombination is said to be additive; when it is beween 1.0 and 0.5, thecombination is defined as subsynergistic, and when it is lower than 0.5,the combination is by defined as synergistic. When the minimum FIC indexis between 1.0 and 2.0, the combination is defined as subantagonisticand, when it is higher than 2.0, the combination is defined asantagonistic.

The pharmaceutical composition or combined preparation with synergisticactivity against viral infection according to this invention may containthe pteridine compound of formula (II) over a broad content rangedepending on the contemplated use and the expected effect of thepreparation. Generally, the pteridine content of the combinedpreparation is within the range of 0.1 to 99.9% by weight, preferablyfrom 1 to 99% by weight, more preferably from 5 to 95% by weight.

The pharmaceutical compositions and combined preparations according tothis invention may be administered orally or in any other suitablefashion. Oral administration is preferred and the preparation may havethe form of a tablet, aqueous dispersion, dispersable powder or granule,emulsion, hard or soft capsule, syrup, elixir or gel. The dosing formsmay be prepared using any method known in the art for manufacturingthese pharmaceutical compositions and may comprise as additivessweeteners, flavoring agents, coloring agents, preservatives and thelike. Carrier materials and excipients are detailed hereinbelow and mayinclude, inter alia, calcium carbonate, sodium carbonate, lactose,calcium phosphate or sodium phosphate; granulating and disintegratingagents, binding agents and the like. The pharmaceutical composition orcombined preparation of this invention may be included in a gelatincapsule mixed with any inert solid diluent or carrier material, or hasthe form of a soft gelatin capsule, in which the ingredient is mixedwith a water or oil medium. Aqueous dispersions may comprise thebiologically active composition or combined preparation in combinationwith a suspending agent, dispersing agent or wetting agent. Oildispersions may comprise suspending agents such as a vegetable oil.Rectal administration is also applicable, for instance in the form ofsuppositories or gels. Injection (e.g. intramuscularly orintraperiteneously) is also applicable as a mode of administration, forinstance in the form of injectable solutions or dispersions, dependingupon the disorder to be treated and the condition of the patient.

Auto-immune disorders to be prevented or treated by the pharma-ceuticalcompositions or combined preparations of this invention include bothsystemic auto-immune diseases such as but not limited to lupuserythematosus, psoriasis, vasculitis, polymyositis, scleroderma,multiple sclerosis, ankylosing spondilytis, rheumatoid arthritis andSjögren syndrome; auto-immune endocrine disorders such as thyroiditis;and organ-specific auto-immune diseases such as but not limited toAddison disease, hemolytic or pernicious anemia, Goodpasture syndrome,Graves disease, idiopathic thrombocytopenic purpura, insulin-dependentdiabetes mellitus, juvenile diabetes, uveitis, Crohn's disease,ulcerative colitis, pemphigus, atopic dermatitis, autoimmune hepatitis,primary biliary cirrhosis, auto-immune pneumonitis, autoimmune carditis,myasthenia gravis, glomerulonephritis and spontaneous infertility.

Transplant rejections to be prevented or treated by the pharmaceuticalcompositions or combined preparations of this invention include therejection of transplanted or grafted organs or cells (both allograftsand xenografts), such as but not limited to host versus graft reactiondisease. The term “organ” as used herein means all organs or parts oforgans in mammals, in particular humans, such as but not limited tokidney, lung, bone marrow, hair, cornea, eye (vitreous), heart, heartvalve, liver, pancreas, blood vessel, skin, muscle, bone, intestine orstomach. “Rejection” as used herein mean all reactions of the recipientbody or of the transplanted organ which in the end lead to cell or issuedeath in the transplanted organ or adversely affect the functionalability and viability of the transplanted organ or the recipient. Inparticular, this means acute and chronic rejection reactions. Alsoincluded in this invention is preventing or treating the rejection ofcell transplants and xenotransplantation. The major hurdle forxenotransplantation is that even before the T lymphocytes, responsiblefor the rejection of allografts, are activated, the innate immunesystem, especially T-independent B lymphocytes and macrophages areactivated. This provokes two types of severe and early acute rejectioncalled hyper-acute rejection and vascular rejection, respectively. Thepresent invention addresses the problem that conventionalimmunosuppressant drugs like cyclosporin A are ineffective inxenotransplantation. The ability of the compounds of this invention tosuppress T-independent xeno-antibody production as well as macrophageactivation may be evaluated in the ability to prevent xenograftrejection in athymic, T-deficient mice receiving xenogenic hamster-heartgrafts.

Cell proliferative disorders to be prevented or treated by thepharmaceutical compositions or combined preparations of this inventioninclude any kind of tumor progression or invasion or metastasisinhibition of a cancer, preferably one selected from the groupconsisting of lung cancer, leukaemia, ovarian cancer, sarcoma, Kaposi'ssarcoma, meningioma, colon cancer, lymp node tumor, glioblastomamultiforme, prostate cancer or skin carcinose.

CNS disorders to be prevented or treated by the pharmaceuticalcompositions of this invention include cognitive pathologies such asdementia, cerebral ischemia, trauma, epilepsy, schizophrenia, chronicpain and neurologic disorders such as but not limited to depression,social phobia and obsessive compulsive disorders.

Cardiovascular disorders to be prevented or treated by thepharmaceutical compositions of this invention include ischemicdisorders, infarct or reperfusion damage, atherosclerosis and stroke.

Allergic conditions to be prevented or treated by the pharmaceuticalcompositions of this invention include those caused by the pollen ofgraminae, the presence of pets, as well as more severe forms, such asasthma, characterized by inflammation of airways and bronchospasm.Without wishing to be bound by theory, the antiallergic effect of thecompounds of the invention may be related to their suppression ofcertain B-cell activation pathways, which can lead to the suppression ofIgE release. It may also be related to their properties of inhibitingcertain Th2 cytokines, such as IL-5, IL-13 or IL-10, involved in asthma.

The term a “pharmaceutically acceptable carrier or excipient” as usedherein in relation to pharmaceutical compositions and combinedpreparations means any material or substance with which the activeprinciple, i.e. the pteridine derivative of formula (II), and optionallythe immunosuppressant or immunomodulator or antineoplastic drug orantiviral agent, may be formulated in order to facilitate itsapplication or dissemination to the locus to be treated, for instance bydissolving, dispersing or diffusing the said composition, and/or tofacilitate its storage, transport or handling without impairing itseffectiveness. The pharmaceutically acceptable carrier may be a solid ora liquid or a gas which has been compressed to form a liquid, i.e. thecompositions of this invention can suitably be used as concentrates,emulsions, solutions, granulates, dusts, sprays, aerosols, pellets orpowders.

Suitable pharmaceutical carriers for use in the said pharmaceuticalcompositions and their formulation are well known to those skilled inthe art. There is no particular restriction to their selection withinthe present invention although, due to the usually low or very lowwater-solubility of the pteridine derivatives of this invention, specialattention will be paid to the selection of suitable carrier combinationsthat can assist in properly formulating them in view of the expectedtime release profile. Suitable pharmaceutical carriers include additivessuch as wetting agents, dispersing agents, stickers, adhesives,emulsifying or surface-active agents, thickening agents, complexingagents, gelling agents, solvents, coatings, antibacterial and antifungalagents (for example phenol, sorbic acid, chlorobutanol), isotonic agents(such as sugars or sodium chloride) and the like, provided the same areconsistent with pharmaceutical practice, i.e. carriers and additiveswhich do not create permanent damage to mammals. The pharmaceuticalcompositions of the present invention may be prepared in any knownmanner, for instance by homogeneously mixing, dissolving, spray-drying,coating and/or grinding the active ingredients, in a one-step or amulti-steps procedure, with the selected carrier material and, whereappropriate, the other additives such as surface-active agents. may alsobe prepared by micronisation, for instance in view to obtain them in theform of microspheres usually having a diameter of about 1 to 10 μm,namely for the manufacture of microcapsules for controlled or sustainedrelease of the biologically active ingredient(s).

Suitable surface-active agents to be used in the pharmaceuticalcompositions of the present invention are non-ionic, cationic and/oranionic materials having good emulsifying, dispersing and/or wettingproperties. Suitable anionic surfactants include both water-solublesoaps and water-soluble synthetic surface-active agents. Suitable soapsare alkaline or alkaline-earth metal salts, unsubstituted or substitutedammonium salts of higher fatty acids (C₁₀-C₂₂), e.g. the sodium orpotassium salts of oleic or stearic acid, or of natural fatty acidmixtures obtainable form coconut oil or tallow oil. Syntheticsurfactants include sodium or calcium salts of polyacrylic acids; fattysulphonates and sulphates; sulphonated benzimidazole derivatives andalkylarylsulphonates. Fatty sulphonates or sulphates are usually in theform of alkaline or alkaline-earth metal salts, unsubstituted ammoniumsalts or ammonium salts substituted with an alkyl or acyl radical havingfrom 8 to 22 carbon atoms, e.g. the sodium or calcium salt oflignosulphonic acid or dodecylsulphonic acid or a mixture of fattyalcohol sulphates obtained from natural fatty acids, alkaline oralkaline-earth metal salts of sulphuric or sulphonic acid esters (suchas sodium lauryl sulphate) and sulphonic acids of fatty alcohol/ethyleneoxide adducts. Suitable sulphonated benzimidazole derivatives preferablycontain 8 to 22 carbon atoms. Examples of alkylarylsulphonates are thesodium, calcium or alcanolamine salts of dodecylbenzene sulphonic acidor dibutyl-naphtalenesulphonic acid or a naphtalene-sulphonicacid/formaldehyde condensation product. Also suitable are thecorresponding phosphates, e.g. salts of phosphoric acid ester and anadduct of p-nonylphenol with ethylene and/or propylene oxide, orphospholipids. Suitable phospholipids for this purpose are the natural(originating from animal or plant cells) or synthetic phospholipids ofthe cephalin or lecithin type such as e.g. phosphatidylethanolamine,phosphatidylserine, phosphatidylglycerine, lysolecithin, cardiolipin,dioctanyl-phosphatidylcholine, dipalmitoylphoshatidylcholine and theirmixtures.

Suitable non-ionic surfactants include polyethoxylated andpolypropoxylated derivatives of alkylphenols, fatty alcohols, fattyacids, aliphatic amines or amides containing at least 12 carbon atoms inthe molecule, alkylarenesulphonates and dialkylsulphosuccinates, such aspolyglycol ether derivatives of aliphatic and cycloaliphatic alcohols,saturated and unsaturated fatty acids and alkylphenols, said derivativespreferably containing 3 to 10 glycol ether groups and 8 to 20 carbonatoms in the (aliphatic) hydrocarbon moiety and 6 to 18 carbon atoms inthe alkyl moiety of the alkylphenol. Further suitable non-ionicsurfactants are water-soluble adducts of polyethylene oxide withpoylypropylene glycol, ethylenediaminopolypropylene glycol containing 1to 10 carbon atoms in the alkyl chain, which adducts contain 20 to 250ethyleneglycol ether groups and/or 10 to 100 propyleneglycol ethergroups. Such compounds usually contain from 1 to 5 ethyleneglycol unitsper propyleneglycol unit. Representative examples of non-ionicsurfactants are nonylphenol-polyethoxyethanol, castor oil polyglycolicethers, polypropylene/polyethylene oxide adducts,tributylphenoxypolyethoxyethanol, polyethyleneglycol andoctylphenoxypolyethoxyethanol. Fatty acid esters of polyethylenesorbitan (such as polyoxyethylene sorbitan trioleate), glycerol,sorbitan, sucrose and pentaerythritol are also suitable non-ionicsurfactants.

Suitable cationic surfactants include quaternary ammonium salts,preferably halides, having 4 hydrocarbon radicals optionally substitutedwith halo, phenyl, substituted phenyl or hydroxy; for instancequaternary ammonium salts containing as N-substituent at least oneC₈-C₂₂ alkyl radical (e.g. cetyl, lauryl, palmityl, myristyl, oleyl andthe like) and, as further substituents, unsubstituted or halogenatedlower alkyl, benzyl and/or hydroxy-lower alkyl radicals.

A more detailed description of surface-active agents suitable for thispurpose may be found for instance in “McCutcheon's Detergents andEmulsifiers Annual” (MC Publishing Crop., Ridgewood, N.J., 1981),“Tensid-Taschenbuch”, 2^(nd) ed. (Hanser Verlag, Vienna, 1981) and“Encyclopaedia of Surfactants (Chemical Publishing Co., New York, 1981).

Structure-forming, thickening or gel-forming agents may be included intothe pharmaceutical compositions and combined preparations of theinvention. Suitable such agents are in particular highly dispersedsilicic add, such as the product commercially available under the tradename Aerosil; bentonites; tetraalkyl ammonium salts of montmorillonites(e.g., products commercially available under the trade name Bentone),wherein each of the alkyl groups may contain from 1 to 20 carbon atoms;cetostearyl alcohol and modified castor oil products (e.g. the productcommercially available under the trade name Antisettle).

Gelling agents which may be included into the pharmaceuticalcompositions and combined preparations of the present invention include,but are not limited to, cellulose derivatives such ascarboxymethylcellulose, cellulose acetate and the like; natural gumssuch as arabic gum, xanthum gum, tragacanth gum, guar gum and the like;gelatin; silicon dioxide; synthetic polymers such as carbomers, andmixtures thereof. Gelatin and modified celluloses represent a preferredclass of gelling agents.

Other optional excipients which may be included in the pharmaceuticalcompositions and combined preparations of the present invention includeadditives such as magnesium oxide; azo dyes; organic and inorganicpigments such as titanium dioxide; UV-absorbers; stabilisers; odormasking agents; viscosity enhancers; antioxidants such as, for example,ascorbyl palmitate, sodium bisulfite, sodium metabisulfite and the like,and mixtures thereof; preservatives such as, for example, potassiumsorbate, sodium benzoate, sorbic add, propyl gallate, benzylalcohol,methyl paraben, propyl paraben and the like; sequestering agents such asethylene-diamine tetraacetic acid; flavoring agents such as naturalvanillin; buffers such as citric acid and acetic acid; extenders orbulking agents such as silicates, diatomaceous earth, magnesium oxide oraluminum oxide; densification agents such as magnesium salts; andmixtures thereof.

Additional ingredients may be included in order to control the durationof action of the biologically-active ingredient in the compositions andcombined preparations of the invention. Control release compositions maythus be achieved by selecting appropriate polymer carriers such as forexample polyesters, polyamino-acids, polyvinyl-pyrrolidone,ethylene-vinyl acetate copolymers, methylcellulose,carboxymethylcellulose, protamine sulfate and the like. The rate of drugrelease and duration of action may also be controlled by incorporatingthe active ingredient into particles, e.g. microcapsules, of a polymericsubstance such as hydrogels, polylactic acid, hydroxymethyl-cellulose,polymethyl methacrylate and the other above-described polymers. Suchmethods include colloid drug delivery systems like liposomes,microspheres, microemulsions, nanoparticles, nanocapsules and so on.Depending on the route of administration, the pharmaceutical compositionor combined preparation of the invention may also require protectivecoatings.

Pharmaceutical forms suitable for injectable use include sterile aqueoussolutions or dispersions and sterile powders for the extemporaneouspreparation thereof. Typical carriers for this purpose therefore includebiocompatible aqueous buffers, ethanol, glycerol, propylene glycol,polyethylene glycol, complexing agents such as cyclodextrins and thelike, and mixtures thereof.

Since, in the case of combined preparations including the pteridinederivative of this invention and an immunosuppressant or immunomodulatoror antihistamine or antineoplastic drug or antiviral agent, bothingredients do not necessarily bring out their synergistic therapeuticeffect directly at the same time in the patient to be treated, the saidcombined preparation may be in the form of a medical kit or packagecontaining the two ingredients in separate but adjacent form. In thelatter context, each ingredient may therefore be formulated in a waysuitable for an administration route different from that of the otheringredient, e.g. one of them may be in the form of an oral or parenteralformulation whereas the other is in the form of an ampoule forintravenous injection or an aerosol.

The present invention further relates to a method for preventing ortreating a disease selected from the group consisting of CNS disorders,cell proliferative disorders, allergic conditions, viral infections,immune and auto-immune disorders and transplant rejections in a subjector patient by administering to the patient in need thereof an effectiveamount of a pteridine derivative having the general formula (II),optionally together with an effective amount of anotherimmunosuppressant or immunomodulator or antineoplastic drug or antiviralagent, or a pharmaceutical composition such as disclosed above inextensive details. The effective amount is usually in the range of 0.01mg to 20 mg, preferably 0.1 mg to 5 mg, per day per kg bodyweight forhumans. Depending upon the pathologic condition to be treated and thepatient's condition, the said effective amount may be divided intoseveral sub-units per day or may be administered at more than one dayintervals. The patient to be treated may be any warm-blooded animal,preferably a human being, suffering from said pathologic condition.

The following examples are intended to illustrate and not to limit thescope of the present invention in all its aspects.

EXAMPLE 1 Synthesis of 2,6-diamino-4-ethoxy-pyrimidine

To a solution of sodium (1.05 g) in ethanol (50 ml) was added4-chloro-2,6-diaminopyrimidine (6 g, 41.4 mmoles). The resultingsolution was heated in a reactor for 6 hours at 160° C. The reactionmixture was cooled down and the precipitated sodium chloride wasfiltered off. The filtrate was concentrated and precipitated fromethanol (two times), affording the pure title compound as a white solid(4.53 g, 72% yield). The spectral data are identical to those describede.g. by W. Pfleiderer et al. in Chem. Ber. (1961) 94, 12.

EXAMPLE 2 Synthesis of 2,6-diamino-4-isopropoxy-pyrimidine

The same procedure as in example 1 was followed using isopropanolinstead of ethanol. The filtrate was pure enough for further reactionwithout purification. The spectral data are identical to those describede.g. by W. Pfleiderer et al. in Chem. Ber. (1961) 94, 12.

EXAMPLE 3 Synthesis of 5-nitroso-2,6-diamino-4-ethoxy-pyrimidine

To a solution of the compound of example 51 (6.13 g, 39.8 mmoles) in 20%aqueous acetic acid (57 ml) was added dropwise a solution of NaNO₂ (3.29g) in water (13 ml) at 80° C. A pink precipitate was formed and stirredat 80° C. for an additional 2 hours. The reaction mixture was cooleddown in the refrigerator overnight and the resulting precipitate wasfiltered off, yielding the title compound as a pink powder (4.98 g,yield 68%). Spectral data are identical with those described e.g. by W.Pfleiderer et al. in Chem. Ber. (1961) 94, 12.

EXAMPLE 4 Synthesis of 5-nitroso-2,6-diamino-4-isopropoxy-pyrimidine

The same procedure was followed as in example 3 but starting from thecompound of example 2. The product has identical spectral data to thosedescribed by W. Pfleiderer et al. (cited supra).

EXAMPLE 5 Synthesis of 2,5,6-triamino-4-ethoxy-pyrimidine

To a suspension of the compound of example 3 (7.12 g, 38.9 mmoles) inwater (150 ml) at 60° C. was added sodium dithionite (46.7 mmol, 8.12g). Additional sodium dithionite was added till the pink colourcompletely dis-appeared and a yellow solution was formed. The solutionwas stirred at 60° C. for another 4 hours. Water was evaporated and theresulting residue was precipitated from a small amount of water,providing the title compound as a yellow powder (4.02 g, yield 61%).Spectral data are identical with literature data (W. Pfleiderer et al.cited supra).

EXAMPLE 6 Synthesis of 2,5,6-triamino-4-isopropoxy-pyrimidine

The procedure of example 5 was followed, however using the compound ofexample 4 as the starting material. The spectral data of the productobtained are identical with the literature data (W. Pfleiderer et al.cited supra).

EXAMPLE 7 Synthesis of 2-amino-4-ethoxy-pteridin

To a solution of 2,5,6-triamino-4-ethoxy-pyrimidine (10.54 g, 62.37mmoles) in ethanol (160 ml) was added glyoxal (40% solution in water,2.7 ml, 18.6 mmoles). The reaction mixture was refluxed for 4 hours.Some insoluble material was filtered off. The filtrate was concentratedin vacuo and the residue purified by flash chromatography (silica, usinga CH₃OH/CH₂Cl₂ mixture (5:95) as the eluent), providing the pure titlecompound (7.34 g, yield: 62%). The spectral data of the product areidentical with the literature data (W. Pfleiderer et al. cited supra).

EXAMPLE 8 Synthesis of 2-amino-4-isopropoxy-pteridin

The procedure of example 7 was repeated, however using isopropanol asthe solvent instead of ethanol. The spectral data of the productobtained are identical with the literature data (W. Pfleiderer et al.cited supra).

EXAMPLE 9 Synthesis of 2-amino-4-ethoxypteridine-N⁸-oxide

To a cooled (0° C.) solution of the compound of example 7 (2.47 g, 12.9mmoles) in trifluoroacetic acid (53 ml) was added dropwise 2.53 ml of a35% aqueous H₂O₂ solution. The reaction mixture was kept at 4° C. fortwo days in the refrigerator, whereby another 1.25 ml of the same H₂O₂solution was added after 1 day. The solution was concentrated in vacuo.The residue was suspended in water and neutralized by the addition of aconcentrated ammonia solution. Evaporation of the solvent in vacuo andpurification of the residue by flash chromatography (silica, using aCH₃OH/CH₂Cl₂ mixture (6:94) as the eluent) provided the title compoundas a yellow powder (861 mg, yield: 32%). Mass spectrum data are asfollows: m/z (%): 230 ([M+Na]⁺, 30), 208 ([M+H]⁺, 100), 180[(M+H-ethene)⁺, 10].

EXAMPLE 10 Synthesis of 2-amino-4-isopropoxypteridine-N⁸-oxide

The procedure as described in example 9 was followed, however using thecompound of example 61 as the starting material. Mass spectrum data areas follows: m/z (%): 222 ([M+H]⁺, 100), 180 ([M+H-propene]⁺, 60).

EXAMPLE 11 Synthesis of 2-amino-6-chloro-4-ethoxypteridine

A suspension of the compound of example 9 (460 mg, 2.22 mmoles) inacetyl chloride (5.5 ml) was stirred at −40° C. Trifluoroacetic acid(1.69 ml) was then added dropwise. The resulting solution was slowlywarmed up to 0° C. and stirred for an additional 4 hours at 0° C.Reaction was carefully quenched with ice, followed by neutralizationwith a concentrated ammonia solution (pH=8). The aqueous phase wasextracted with CH₂Cl₂ (five times). The combined organic layers wereconcentrated in vacuo and the residue was purified by flashchromatography (silica, using a CH₃OH/CH₂Cl₂ mixture (1:99) as theeluent), thus providing the title compound as a yellow powder (360 mg,yield: 72%). This compound was further characterized as follows:

mass spectrum: m/z (%): 226 ([M+H]⁺, 100), ¹H-NMR (200 MHz, DMSO-d₆): δ1.42 (3 H, t), 4.52 (2 H, q), 7.42 (2 H, d) and 8.85 (1H, s) ppm,¹³C-NMR (50 MHz, DMSO-d₆): δ 14.19, 63.58, 121.74, 140.22, 150.99,156.13, 161.98 and 165.97 ppm.

EXAMPLE 12 Synthesis of 2-amino-6-chloro-4-isopropoxypteridine

The procedure as described in example 11 was followed, however startingfrom the compound of example 63. The mass spectrum data of the resultingcompound are as follows: m/z (%): 240 ([M+H]⁺, 55), 198 ([M+H-propene]⁺,100).

EXAMPLES 13 TO 30 Synthesis of 2-amino-6-aryl-4-ethoxypteridines and2-amino-6-heteroaryl-4-ethoxypteridines

The general procedure used for preparing2-amino-6-aryl-4-ethoxy-pteridines is as follows: to a degassed solutionof the compound of example 64 (50 mg, 0.22 mmole) in THF (5 ml) wasadded a degassed solution of sodium carbonate (5 ml of a 0.4 M solutionin water), tetrakis(triphenyl-phosphine) palladium (0.013 mmole, 14 mg)and an arylboronic or (examples 72 and 73) heteroarylboronic acid (0.22mmole). The solution was refluxed for 4 hours. Solvents wereconcentrated in vacuo and the residue was purified by flashchromatography (silica) with an appropriate CH₃OH/CH₂Cl₂ mixture (2:98or 3:97) as the eluent (except for the compound of example 82, which waseluted with an acetone/CH₂Cl₂ (7:3) mixture). This procedure provided,with a yield ranging from 16% to 60% depending upon the aryl orheteroaryl group (from the arylboronic or heteroarylboronic acid)introduced at the 6-position of the pteridine ring, the following purefinal compounds which were characterized by their mass spectrum MS andoptionally by their ¹H-NMR (200 MHz, DMSO-d₆) spectrum:

-   2-amino-6-(p-methoxyphenyl)-4-ethoxy-pteridine (example 13): MS 298    ([M+H]⁺, 100), 270 ([M+H-ethene]⁺, 55);-   2-amino-6-(o-methoxyphenyl)-4-ethoxy-pteridine (example 14): MS 298    ([M+H]⁺, 100), 270 ([M+H-ethene]⁺, 30);-   2-amino-6-(m-methoxyphenyl)-4-ethoxy-pteridine (example 15): MS 298    ([M+H]⁺, 100), 270 ([M+H-ethene]⁺, 35); ¹H-NMR: 1.46 (3 H, t), 3.85    (3 H, s), 4.58 (2 H, q), 7.06 (1 H, dd), 7.33 (2 H, br s), 7.46 (1    H, t), 7.68 (1 H, m) and 9.43 (1 H, s) ppm;-   2-amino-6-(3,4-difluorophenyl-4-ethoxy-pteridine (example 16): MS    304 ([M+H]⁺, 100), 270 ([M+H-ethene]⁺, 35); ¹H-NMR: 1.45 (3 H, t),    4.57 (2 H, q), 7.42 (2 H, br s), 7.60 (1 H, q), 7.98 (1 H, d), 8.16    (1 H, t) and 9.42 (1 H, s) ppm;-   2-amino-6-(p-dimethylaminophenyl)-4-ethoxy-pteridine (example 17) MS    311 ([M+H]⁺, 100), 283 ([M+H-ethene]⁺, 35);-   2-amino-6-(p-trifluoromethylphenyl)-4-ethoxy-pteridine (example 18):    MS 336 ([M+H]⁺, 100), 308 ([M+H-ethene]⁺, 50);-   2-amino-6-(2-thienyl)-4-ethoxy-pteridine (example 19): MS 274    ([M+H]⁺, 100), 246 ([M+H-ethene]⁺, 40);-   2-amino-6-(3-thienyl)-4-ethoxy-pteridine (example 20): MS 274    ([M+H]⁺, 100), 246 ([M+H-ethene]⁺, 45);-   2-amino-6-(3,4-dichlorophenyl)-4-ethoxy-pteridine (example 21): MS    337 ([M+H]⁺, 100); ¹H-NMR: 1.46 (3 H, t), 4.59 (2 H, q), 7.42 (2 H,    br s), 7.81 (1 H, d), 8.14 (1 H, dd), 8.37 (1 H, d) and 9.47 (1H, s)    ppm;-   2-amino-6-(p-cyanophenyl)-4-ethoxy-pteridine (example 22): MS 293    ([M+H]⁺, 100), 265 ([M+H-ethene]⁺, 65);-   2-amino-6-(p-ethoxyphenyl)-4-ethoxy-pteridine (example 23): MS 312    ([M+H]⁺, 100), 284 ([M+H-ethene]⁺, 70);-   2-amino-6-(p-fluorophenyl)-4-ethoxy-pteridine (example 24): MS 286    ([M+H]⁺, 100), 258 ([M+H-ethene)⁺, 45);-   2-amino-6-(p-ethylphenyl)-4-ethoxy-pteridine (example 25): MS 296    ([M+H]⁺, 100), 268 ([M+H-ethene)⁺, 45);-   2-amino-6-(p-acetylphenyl)-4-ethoxy-pteridine (example 26): MS 310    ([M+H]⁺, 100), 282 ([M+H-ethene]⁺, 60);-   2-amino-6-(3-methyl-4-fluorophenyl)-4-ethoxy-pteridine (example 27):    MS 300 ([M+H]⁺, 100), 272 ([M+H-ethene]⁺, 30);-   2-amino-6-(p-thiomethylphenyl)-4-ethoxy-pteridine (example 28): MS    314 ([M+H]⁺, 100), 286 ([M+H-ethene]⁺, 35);-   2-amino-6-(p-N,N-dimethylbenzamido)-4-ethoxy-pteridine (example 29)    MS 338 ([M+H]⁺, 100), 311 ([M+H-ethene]⁺, 15); and-   2-amino-6-(3,4-dimethoxyphenyl)-4-ethoxy-pteridine (example 30): MS    328 ([M+H]⁺, 100), 300 ([M+H-ethene]⁺, 40).

EXAMPLES 31 TO 45 Synthesis of 2-amino-6-aryl-4-isopropoxypteridines and2-aminoheteroaryl-4-isopropoxypteridines

The procedure as described in examples 13 to 30 was followed while using2-amino-6-chloro-4-isopropoxypteridine as the starting material, exceptthat longer reaction times were needed (refluxing overnight instead of 4hours). This procedure provided, with a yield ranging from 10% to 70%depending upon the aryl or heteroaryl group introduced at the 6-positionof the pteridine ring, the following pure final compounds which werecharacterized by their mass spectrum:

-   2-amino-6-(3-methyl-4-methoxyphenyl)-4-isopropoxypteridine (example    31): MS 326 ([M+H]⁺, 100), 284 ([M+H-propene]⁺, 30);-   2-amino-6-(3,4-dimethylphenyl)-4-isopropoxypteridine (example 32):    MS 310 ([M+H]⁺, 100), 268 ([M+H-propene]⁺, 60);-   2-amino-6-(3-chloro-4-trifluoromethylphenyl)-4-isopropoxypteridine    (example 33): MS 384 ([M+H]⁺, 20), 342 ([M+H-propene]⁺, 50);-   2-amino-6-(3-chloro-4-fluorophenyl)-4-isopropoxypteridine (example    34): MS 334 ([M+H]⁺, 20), 292 ([M+H-propene]⁺, 50);-   2-amino-6-(p-N,N-diethylbenzamido)-4-isopropoxypteridine (example    35): MS 381 ([M+H]⁺, 100);-   2-amino-6-(p-trifluoromethylphenyl)-4-isopropoxypteridine (example    36): MS 350 ([M+H]⁺, 100), 308 ([M+H-propene]⁺, 30);-   2-amino-6-(3,4-difluorophenyl)-4-isopropoxypteridine (example 37):    MS 318 ([M+H]⁺, 100), 276 ([M+H-propene]⁺, 50);-   2-amino-6-(p-methoxyphenyl-4-isopropoxypteridine (example 38): MS    312 ([M+H]⁺, 100), 270 ([M+H-propene]⁺, 50);-   2-amino-6-(p-ethoxyphenyl)-4-isopropoxypteridine (example 39): MS    326 ([M+H]⁺, 55), 284 ([M+H-propene]⁺, 100);-   2-amino-6-(p-dimethylbenzamido)-4-isopropoxypteridine (example 40):    MS 353 ([M+H]⁺, 75), 311 ([M+H-propene]⁺, 100);-   2-amino-6-(3-thienyl)-4-isopropoxypteridine (example 41): MS 288    ([M+H]⁺, 55), 246 ([M+H-propene]⁺, 100);-   2-amino-6-(p-cyanophenyl)-4-isopropoxypteridine (example 42): MS 307    ([M+H]⁺, 40), 265 ([M+H-propene]⁺, 100);-   2-amino-6-(p-benzoic acid methyl ester)-4-isopropoxypteridine    (example 43): MS 340 ([M+H]⁺, 75), 298 ([M+H-propene]⁺, 100);-   2-amino-6-(p-acetylphenyl)-4-isopropoxypteridine (example 44): MS    324 ([M+H]⁺, 55), 282 ([M+H-propene]⁺, 100); and-   2-amino-6-(3,4-dimethoxyphenyl)-4-isopropoxypteridine (example 45):    MS 342 ([M+H]⁺, 100), 300 ([M+H-propene]⁺, 60).

EXAMPLE 46 Synthesis of 2,6-diamino-5-nitroso-4-hydroxypyrimidine

To a solution of 2,6-diamino-4-hydroxypyrimidine (12.9 g, 102.2 mmoles)in 200 ml of a 10% acetic acid solution in water at 80° C. was addeddropwise a solution of NaNO₂ (7.05 g, 102.2 mmoles) in 20 ml water. Apink precipitate was formed, which was further stirred for 1 hour at 80°C. The reaction mixture was cooled down in the refrigerator overnight.The precipitate was filtered off and dried over P₂O₅, providing thetitle compound as a pink powder (15.43 g, yield: 97%). The spectral dataare in accordance with literature data (Landauer et al. in J. Chem. Soc.(1953) 3721-3722).

EXAMPLE 47 Synthesis of 2,5,6-triamino-4-hydroxypyrimidine

A suspension of the compound of example 46 (15 g, 96.7 mmoles) in anammonium sulfide solution (20% in water, 200 ml) was stirred overnightat 50° C. The reaction mixture was cooled down in the refrigerator andthe precipitate was filtered off, providing the title compound as ayellow powder (11.33 g, yield: 83%). The spectral data are identicalwith literature data (Landauer et al. cited supra).

EXAMPLE 48 Synthesis of 2-amino-6-(3,4-dimethoxyphenyl)pterine

To a boiling solution of the compound of example 47 (2.4 g, 17 mmoles)in methanol (100 ml, with 0.9 N HCl) was added dropwise a solution of3,4-dimethoxyphenylglyoxal mono-oxime (3.8 g, 18 mmoles) in methanol(100 ml). The reaction mixture was heated under reflux for 4 hours. Theprecipitate formed was filtered off, washed with water, then ethanol anddiethyl ether, and dried over P₂O₅ under vacuum, providing the titlecompound as a yellow powder (4.33 g, yield: 85%). This compound wasfurther characterized by the following spectra:

¹H-NMR (500 MHz, TFA): δ 4.11 (3 H, s), 4.07 (3 H, s), 7.21 (1 H, d),7.78 (1 H, dd), 7.81 (1 H, d) and 9.32 (1 H, s) ppm; ¹³C-NMR (125 MHz,TFA): δ 56.39, 56.7, 111.94, 113.21, 123.22, 127.41, 127.91, 145.92,149.39, 150.46, 152.47, 153.15, 155.13 and 161.59 ppm.

EXAMPLE 49 Synthesis of 2-acetylamino-6-(3,4-dimethoxyphenyl)pterine

A suspension of the compound of example 48 (10.46 g, 35 mmoles) inacetic anhydride (600 ml) and acetic acid (200 ml) was refluxed for 1hour until a clear solution was formed. By cooling down the reactionmixture in the refrigerator, the precipitate formed was filtered off,washed with ethyl acetate and diethyl ether, and then dried over P₂O₅under vacuum, providing the title compound as a yellow powder (9.19 g,yield: 77%). This compound was further characterized by the followingspectra:

MS: m/z (%): 300 ([M+H]⁺, 100); ¹H-NMR (200 MHz, DMSO-d₆): δ 2.22 (3 H,s), 3.84 (3 H, s), 3.87 (3 H, s), 7.14 (1 H, d), 7.75 (2 H, m) and 9.51(1 H, s) ppm.

EXAMPLE 50 Synthesis of2-acetylamino-4-(1,2,4-triazolyl)-6-(3,4-dimethoxy-phenyl)pteridine

To a solution of phosphorus oxychloride (1.68 ml, 18 mmoles) and1,2,4-triazole (4.96 g, 72 mmoles) in dry pyridine (110 ml) was addedthe compound of example 49 (2.45 g, 7.18 mmoles). The suspension wasstirred at room temperature for 4 hours. The precipitate was filteredoff, washed with pyridine, toluene and diethyl ether. The resultingsolid was dried over P₂O₅ under vacuum, providing the title compound asa yellow powder (2 g, yield: 80%) which afforded the following massspectrum: 392 ([M+H]⁺, 100).

EXAMPLES 51 AND 52 Synthesis of2-amino-4-mercaptoethyl-6-(3,4-dimethoxyphenyl)pteridine and2-amino-4-mercaptoisopropyl-6-(3,4-dimethoxyphenyl) pteridine

To a suspension of the compound of example 50 (0.25 mmole, 100 mg) indioxane (5 ml) was added 1 mmole of either ethanethiol (example 51) orisopropanethiol (example 52) and sodium (12 mg, 0.5 mmole). Thesuspension was stirred for 24 hours at room temperature. The solvent wasconcentrated in vacuo and the residue purified by flash chromatography(silica, using a CH₃OH/CH₂Cl₂ mixture (5:95) as an eluent), followed bypurification by preparative TLC, providing the pure title compounds asyellow powders with yields ranging from 20 to 30%. Both compounds werecharacterized by their mass spectrum as follows:

-   2-amino-4-mercaptoethyl-6-(3,4-dimethoxyphenyl) pteridine: 344    ([M+H]⁺, 100);-   2-amino-4-mercaptoisopropyl-6-(3,4-dimethoxyphenyl) pteridine: 357    ([M+H]⁺, 100).

EXAMPLE 53 Synthesis of a Mixture of 2,4-diamino-6-(p-methoxyphenyl)pteridine and 2,4-diamino-7-(p-methoxyphenyl)pteridine

2,4,5,6-tetra-aminopyrimidine (10 mmoles, 1.4 g) was dissolved in water(50 ml) and the pH was adjusted to 9 with ammonium hydroxide. A solutionof 4-methoxyphenylglyoxal (11 mmoles, 1.8 g) in ethanol (10 ml) wasadded dropwise and the solution was refluxed for 1 hour. The yellowprecipitate formed was filtered off and washed with water, ethanol anddiethyl ether. NMR analysis reveals the obtention of a mixture (1.2 g,45% yield) consisting of 87% of 2,4-diamino-7-(p-methoxyphenyl)pteridineand 13% of 2,4-diamino-7-(p-methoxyphenyl)pteridine. ¹H-NMR (500 MHz,TFA): δ 4.04 (3 H, s), 4.08 (3 H, s), 7.15 (2 H, d), 7.25 (2 H, d), 8.19(2 H, d), 8.30 (2 H, d), 9.27 (1 H, s) and 9.37 (1 H, s) ppm.

EXAMPLE 54 Synthesis of a Mixture of 2-amino-6-(p-methoxyphenyl)pterinand 2-amino-7-(p-methoxyphenyl)pterin

The mixture obtained in example 53 (1.2 g, 4.5 mmoles) was suspended inNaOH 1 N (80 ml) and refluxed till a solution was obtained. The hotsolution was treated with acetic acid till pH 5, then cooled down andthe resulting precipitate was filtered off and washed with water,ethanol and diethyl ether, providing a mixture of2-amino-6-(p-methoxyphenyl)pterin and 2-amino-7-(p-methoxyphenyl) pterinas a yellow powder (1 g, yield: 82%). Mass spectrum: 270 ([M+H]⁺, 100).

EXAMPLE 55 Synthesis of 2-acetylamino-6-(D-methoxyphenyl)pterin and2-acetylamino-7-(p-methoxyphenyl)pterin

A suspension of the mixture obtained in example 54 (7.43 mmoles, 2 g)was suspended in a mixture of acetic anhydride (50 ml) and acetic acid(50 ml). The suspension was refluxed for 4 hours till a clear solutionwas obtained. Some insoluble material was filtered off and the solutionwas partly evaporated till precipitation starts. Further precipitationwas achieved overnight in the refrigerator. The resulting precipitatewas filtered off and washed with ethyl acetate and diethyl ether,providing a mixture of 2-acetylamino-6-(p-methoxyphenyl)pterin and2-acetylamino-7-(p-methoxyphenyl)pterin as a yellow powder (2.1 g, 91%yield). Mass spectrum: 312 ([M+H]⁺, 100).

EXAMPLE 56 Synthesis of2-acetylamino-4-(1,2,4-triazolyl)-6-(p-methoxy-phenyl)pteridine and2-acetylamino-(1,2,4-triazolyl)-7-(p-methoxyphenyl)pteridine

To a suspension of the mixture obtained in example 55 (1.5 g, 4 mmoles)in dry pyridine (100 ml) was added 1,2,4-triazole (830 mg, 12 mmoles)and 4-chlorophenyl phosphorodichloridate (1 ml, 6 mmoles). Thesuspension was stirred for 2 days at room temperature under nitrogen.The solvents were removed in vacuo. The solid material was suspended indichloromethane and washed with 2% HCl. Evaporation of the solventsprovided a mixture of2-acetylamino-4-(1,2,4-triazolyl)-6-(p-methoxyphenyl) pteridine and2-acetylamino-4-(1,2,4-triazolyl)-7-(p-methoxyphenyl)pteridine.

EXAMPLE 57 Synthesis of2-amino-4-isopropoxy-7-(p-methoxyphenyl)pteridine

To a suspension of the mixture obtained in example 56 (180 mg, 0.50mmole) in isopropanol (8 ml) was added sodium (23 mg, 1 mmole). Thesuspension was stirred at room temperature overnight. The solvents wereevaporated and the residue was purified by preparative TLC (silica,using a methanol/CH₂Cl₂ (7:93) mixture as the eluent). At this stage,both regio-isomers obtained were separated, thus providing the puretitle compound as a yellow powder (yield: 45%) which was furthercharacterized by its mass spectrum: 312 ([M+H]⁺, 65), 270([M+H-propene]⁺, 100).

EXAMPLE 58 Synthesis of2-amino-4-isopropoxy-7-(3,4-dimethoxyphenyl)pteridine

The sequence of reactions described in examples 53 to 57 was followed,however starting from 3,4-dimethoxyphenylglyoxal instead of4-methoxyphenylglyoxal in the first step. This provided2-amino-4-isopropoxy-7-(3,4-dimethoxyphenyl) pteridine, a compound whichwas further characterized by its mass spectrum: 342 ([M+H]⁺, 55), 300([M+H-propene]⁺, 75).

EXAMPLE 59 Synthesis of 2-aminoethoxy-7-(3,4-dimethoxyphenyl) pteridine

The sequence of reactions described in examples 53 to 57 was followed,however starting from 3,4-dimethoxyphenylglyoxal instead of4-methoxyphenylglyoxal in the first step, and from ethanol instead ifisopropanol in the last step. This provided2-amino-4-ethoxy-7-(3,4-dimethoxyphenyl) pteridine, a compound which wasfurther characterized as follows:

MS: 328 ([M+H]⁺, 100), 300 ([M+H-ethene]⁺, 40); ¹H-NMR (500 MHz,DMSO-d₆): δ 1.44 (3 H, t), 3.86 (3 H, s), 3.88 (3 H, s), 4.54 q), 7.13(1 H, d), 7.16 (2 H, br s), 7.85 (1 H, d), 7.88 (1 H, dd) and 9.06 (1 H,s) ppm

¹³C-NMR (125 MHz, DMSO-d₆): δ 14.25, 55.67, 55.76, 63.06, 110.39,111.89, 121.13, 121.25, 128.24, 136.87, 149.28, 151.62, 155.82, 156.72,162.03 and 166.70 ppm.

EXAMPLE 60 Synthesis of2-amino-4-methoxy-7-(3,4-dimethoxyphenyl)pteridine

The sequence of reactions described in examples 53 to 57 was followed,however starting from 3,4-dimethoxyphenylglyoxal instead of4-methoxyphenylglyoxal in the first step, and from methanol instead ifisopropanol in the last step. This provided2-amino-4-ethoxy-7-(3,4-dimethoxyphenyl) pteridine, a compound which wasfurther characterized by its mass spectrum: 314 ([M+H]⁺, 100), 300([M+H-methane]⁺, 20).

EXAMPLE 61 Lymphocyte Activation Tests

Pteridine derivatives were first dissolved (10 mM) in dimethylsulfoxide(hereinafter referred as DMSO) and further diluted in culture mediumbefore use for the following in vitro experiments. The commerciallyavailable culture medium consisted of RPMI-1640+10% foetal calf serum(FCS). Some pteridine derivatives described the previous examples weretested in the following lymphocyte activation tests:

Mixed Lymphocyte Reaction

Peripheral blood mononuclear cells (hereinafter referred as PBMC) wereisolated from heparinized peripheral blood by density gradientcentrifugation over Lymphoprep (Nycomed, Maorstua, Norway). AllogeneicPBMC or Eppstein-Barr Virus-transformed human B cells [commerciallyavailable under the trade name RPMI1788 (ATCC name CCL156)] whichstrongly express B7-1 and B7-2 antigens were used as stimulator cellsafter irradiation with 30 Gy. MLR was performed in triplicate wells.After 5 days incubation at 37° C., 1 μCi [³H]-thymidine was added toeach cup. After a further 16 hours incubation, cells were harvested andcounted in a β-counter. Inhibition of proliferation by a compound (drug)described in some of the previous examples was counted using theformula:

${\%\mspace{11mu}{inhibition}} = {\frac{\left( {{cpm} + {drugs}} \right) - \left( {{cpm}\mspace{14mu}{{cult}.\mspace{14mu}{med}}} \right)}{\left( {{cpm} - {drugs}} \right) - \left( {{OD}\mspace{14mu}{{cult}.\mspace{14mu}{med}}} \right)} \times 100}$wherein cpm is the thymidine count per minute. The MLR assay is regardedby those skilled in the art as an in vitro analogue of the transplantrejection since it is based on the recognition of allogeneic majorhistocompatibility antigens on the stimulator leukocytes, by respondinglymphocytes.Assays for CD3 and CD 28

T cells were purified by removing non-T cells. Briefly, monocytes wereremoved by cold agglutination. The resulting lymphoid cells were furtherpurified by a cell enrichment immunocolumn [Cellect Human T commerciallyavailable from Biotex, Edmonton, Alberta, Canada)] by a process ofnegative selection. More than 95% of the B cells were removed with thisprocedure. After depletion, the resulting T cell preparation was highlypurified, i.e. these cells could not be activated by phytohaemagglutinin(PHA) or rIL-2 alone at concentrations capable of stimulating RBMC priorto deletion.

Highly purified T cells (10⁶/ml) were stimulated by immobilized anti-CD3or anti-CD28 monoclonal antibodies (hereinafter referred as mAb) in thepresence of phorbol myristate acetate (hereinafter referred as PMA).Anti-CD3 mAb (available from CLB, Amsterdam, Netherlands) were fixed ona 96-microwell plates by incubating the wells with 50 μl of mAb solution(1/800 dilution in culture medium). For anti-CD28 mAb (available fromCLB, Amsterdam, Netherlands) 50 μl (1/650 dilution in culture medium)was added directly to the wells. Further, a 20 μl PMA (commerciallyavailable from Sigma, St. Louis, Mo., USA) solution (finalconcentration: 0.5 ng/ml) was added. Subsequently, 20 μl of a pteridinederivative described in the previous examples were added by serialdilution in triplicate wells. Finally 100 μl of the T-cell suspension(10⁶/ml) was added. After 48-hour incubation at 37° C. in 5% CO₂, 20 μlof a bromo-deuridine (hereinafter referred as BrdU) 100 μM solution(commercially available as Cell Proliferation Elisa fromBoehringer-Mannheim Belgium) was added to each well. After a furtherovernight incubation, T-cell proliferation was measured using acalorimetric immuno-assay for qualification of cell proliferation basedon the incorporation of BrdU during DNA synthesis. Optical density(hereinafter referred as OD) was measured by a Behring EL311 platereader at 450 nm (reference wavelength: 690 nm). Inhibition ofproliferation by the pteridine derivative (drug) was counted while usingthe formula:

${\%\mspace{11mu}{inhibition}} = {\frac{\left( {{OD} + {drugs}} \right) - \left( {{OD}\mspace{14mu}{{cult}.\mspace{14mu}{med}}} \right)}{\left( {{OD} - {drugs}} \right) - \left( {{OD}\mspace{14mu}{{cult}.\mspace{14mu}{med}}} \right)} \times 100}$Table 1 (wherein ND means not determined) below shows the IC₅₀ valuesfor various pteridine derivatives in the MLR test and in the CD3 andCD28 assays. The IC₅₀ value represents the lowest concentration of thepteridine derivative (expressed in μmole/l) that resulted in a 50%suppression of the MLR or a 50% reduction in T-cell proliferation (forthe CD3 and CD28 assay).

TABLE 1 Example n° MLR CD 3 CD28 15 8.3 20 7.0 16 >10 9.0 4.0 18 10 100.8 20 ND 15 0.7 25 8.2 10 10 30 0.9 0.7 0.7 32 10 ND ND 45 0.4 6.7 1.7ND: not determined

The above data show that, whereas known immunosuppressant drugs like CyAare known to be active only in the CD3 assay, the pteridine derivativesaccording to the present invention were also active in the CD28 assaywhich is Ca²⁺-calmodulin resistant. The CD28 pathway is a so-calledcosignal pathway which is important for inducing energy and eventolerance in T-cells.

EXAMPLE 62 TNF-α and IL-1 β Assays

Peripheral blood mononuclear cells (herein referred as PBMC), inresponse to stimulation by lipopolysaccharide (LPS), a gram-negativebacterial endotoxin, produce various chemokines, in particular humanTNF-α and IL-1 β. Inhibition of the activation of PBMC can therefore bemeasured by the level of suppression of the production of TNF-α or IL-1β by PBMC in response to stimulation by LPS.

Such inhibition measurement was performed as follows: PBMC were isolatedfrom heparinized peripheral blood by density gradient centrifugationover Lymphoprep (commercially available from Nycomed, Norway). LPS wasthen added to the PMBC suspension in complete medium (10⁶ cells/ml) at afinal concentration of 1 μg/ml. The pteridine derivative to be testedwas added at different concentrations (0.1 μM, 1 μM and 10 μM) and thecells were incubated at 37° C. for 72 hours in 5% CO₂. The supernatantswere collected, then TNF-α and/or IL-1 β concentrations were measuredwith respectively an anti-TNF-α antibody or an anti-IL-1 β antibody in asandwich ELISA (Duo Set ELISA human TNFα, commercially available fromR&D Systems, United Kingdom). The calorimetric reading of the ELISA wasmeasured by a Multiskan RC plate reader (commercially available fromThermoLabsystems, Finland) at 450 nm (reference wavelength: 690 nm).Data analysis was performed with Ascent software 2.6. (also fromThermoLabsystems, Finland): a standard curve (recombinant human TNFα)was drawn and the amount (pg/ml) of each sample on the standard curvewas determined.

The % suppression of human TNFα production or human IL-1 β by thepteridine derivatives of the invention (drugs) was calculated using theformula:

${\%\mspace{11mu}{suppression}} = \frac{\begin{matrix}{{{pg}\text{/}{ml}\mspace{14mu}{in}\mspace{14mu}{drugs}} -} \\{{pg}\text{/}{ml}\mspace{14mu}{in}\mspace{14mu}{{cult}.\mspace{11mu}{med}.}}\end{matrix}}{\begin{matrix}{\left( {{pg}\text{/}{ml}\mspace{14mu}{in}\mspace{14mu}{{cult}.\mspace{14mu}{med}.{+ {LPS}}}} \right) -} \\{{pg}\text{/}{ml}\mspace{14mu}{{cult}.\mspace{14mu}{med}.}}\end{matrix}}$

Table 2 below shows the IC₅₀ values (expressed in μM) of the testedpteridine derivatives in the TNF-α assay.

TABLE 2 Example n° TNF-α 6 8.0 10 4.0 12 9.1 13 4.5 14 10.0 15 10.0 198.5 24 8.1 25 8.5 27 6.8 28 10.0 30 6.6 32 6.7 34 7.5 36 9.1 37 6.6 386.2 39 10.0 41 7.6 42 6.3 44 9.1 45 3.5

EXAMPLE 63 Inhibition of the Release of Cytokines Involved in Asthma

2-amino-4-morpholino-6-(3,4-dimethoxyphenyl)pteridine (a compound knownfrom WO 00/39129) was tested, according to experimental procedures wellestablished in the art, in the inhibition of two cytokines, IL-5 andIL-10, known to be involved in the development of asthma and other formsof severe allergic conditions. At the concentration of 0.1 μM/l, i.e.the IC₅₀ value of this derivative in the MLR test, the said derivativeinhibited the release of IL-5 and IL-10 by 70% and 39%, respectively.

EXAMPLE 64 Inhibition of the Metastasis of Melanoma Cells in Mice

C57BL/6 mice were injected with 1.5×10⁶ B16BL/6 melanoma cells and weredivided into 2 groups. Five days after the injection of the tumor cells,the two groups of animals were administered intraperitoneously, threetimes a week for two subsequent weeks, either (treated group) 20 mg/kgof 2-amino-4-morpholino-6-(3,4-dimethoxyphenyl)pteridine (a compoundknown from WO 00/39129) or (control group) the vehicle (5% DMSO inphosphate buffered saline). At the end of the experiment, all survivingtumor-bearing mice were sacrificed and examined in order to assess theabsence or presence of the black metastases in inguinal and/orpara-aortic lymph nodes (macroscopic metastasis). The findings wereconfirmed by histology, and the experiment done twice. The pooledresults of the two experiments show that 80% of animals from the controlgroup had metastases whereas only 33% of animals from the treated grouphad metastases.

EXAMPLE 65 In Vivo Leukocyte Activation and Immunosuppression WholeBlood Assay

In response to non-self antigens (e.g. microbial antigens,allo-antigens, altered self antigens and auto-antigens), immune cellssuch as lymphocytes, NK cells, monocytes/macrophages and dentritic cellsare activated to generate antigen-reactive effector cells. Activatedimmune cells up-regulate a number of pro-inflammatory genes encodingcytokines (e.g. IL-2, IFN-γ, and TNF-α), chemokines (e.g. monocytechemo-attractant protein-1 (MCP-1), macrophage inflammatory protein-1(MIP-1) and RANTES), and cell surface molecules. These leukocyte surfacemolecules may include activation associated molecules (e.g. CD69, thelymphocyte early activation molecule, and CD71, the transferinreceptor), adhesion molecules (e.g. ICAM-1, LFA-1 and L-selectins),co-stimulatory molecules (e.g. CD28, B7-1 and CD40L) and receptors forcytokines and chemokines (e.g. IL-2R and CCR1).

Immunomodulatory therapies are active by interfering with the responsesof the immune system. Reliable, easy, fast and quantitative methods formonitoring the evolution of the leucocyte surface and cytoplasmaphenotypes during activation are critical in evaluating the functionalcompetence of the immune cells (e.g., during HIV infection), as well asthe efficacy of immunoregulatory therapies in order to achieve optimaltherapeutic effects and minimal side effects. Moreover, examination ofdrug action on leukocyte activation is able to provide criticalevaluation of pharmacokinetic and pharmacodynamic effects ofimmunomodulatory agents. Within this context, the following provides auseful in vivo leukocyte activation and immunosuppression whole bloodassay (hereinafter referred as WBA).

Inbred mice (e.g. AKR strain), weighting from 20 to 30 g, werestimulated by intraperitoneal injection with mitogens specific for Tcells (e.g. Concanavalin A, hereinafter referred as ConA, 500 μg permouse), B cells or monocytes/macrophages (e.g. LPS 20 μg per mouse). Thepteridine derivatives of example 30 and, respectively, example 45, wereadministered intraperitoneously (30 mg/kg per day, administered 2 hoursbefore ConA injection). Peripheral blood was collected 24, 36, 48, andrespectively 72 hours after stimulation for immune fluorescence stainingand FACScan analysis.

FACScan analysis proceeded as follows: heparinized whole blood (50 μl)was removed of red blood cells by incubation with a lysing buffer. Theremaining leukocytes were double-stained with phycoerythrin-conjugatedantibodies specific for T cells (e.g. T cell receptor TCR), B cells(e.g. CD45R/B220 (commercially available from Pharmingen) ormonocytes/macrophages (e.g. F4/80) (commer-cially available fromSerotec) in combination with FITC-conjugated antibodies against variouscell surface markers such as CD69 (early activation molecule), CD25(IL-2 receptor), CD71 (transferring receptor), B7-1/2 (ligand for theco-stimulatory molecule CD28) and CD11b (complement receptor 3). Cellswere examined by flow cytometry using CellQuest software (commerciallyavailable from Decton Dickinson).

Peripheral blood T cells expressed an up-regulated level of CD69,representing up to 3% of TCR⁺, CD69⁺ cells respectively 1 and 2 daysfollowing ConA injecton, as compared to baseline levels (0.5%). T cellsfrom mice treated with the pteridine derivatives of examples 30 and 45show a remarkable reduction of this response to 1.6% and 2.4%respectively.

Peripheral blood T cells showed a rapid up-regulation of CD71 frombaseline level (4.8%) to 9.3% and 5.5% after 1 day and 2 days,respectively. The pteridine derivatives of examples 30 and 45 suppressedConA-stimulated CD71 up-regulation on T cells to baseline levels.

Peripheral blood T cells expressed increased levels of IL-2R with up to3% of TCR+, IL-2R+ cells 1 and 2 days following ConA stimulation, withpeak levels after day 2. Administration of the pteridine derivatives ofexamples 30 and 45 effectively suppresses this effect by reducingexpression to 1.5% and 1.7%, respectively.

Up to 6.6% of peripheral blood T cells displayed an up-regulation of theCD134 molecule expression within 2 days following ConA stimulation, i.e.a two-fold increase of baseline levels (2.2%). Treatment with thepteridine derivatives of examples 30 and 45 suppressed this response to3.5% and 4.2%, respectively.

This FACScan-based cell surface phenotype analysis for in vivo leukocyteactivation, namely for an in vivo whole blood assay, demonstrates theimmunosuppressive effects of the pteridine derivatives of examples 30and 45 in vivo. This observation is consistent with the aboveexperimental results in vitro showing that both compounds inhibit T cellactivation and proliferation in mixed lymphocyte culture, and in the CD3and CD28 assays.

In vivo WBA by injecting immune activators, followed by FACS analysis ofleukocyte surface phenotypes in the whole blood can be applied tomonitor in vivo activation of different types of leukocytes such as Tcells, B cells, monocytes/macrophages, and dentritic cells. Thistechnology allows simple, rapid, direct, stable and quantitativeevaluation of the immune competence of the said leukocytes, as well aspharmacokinetic and pharmacodynamic profiles of the pteridinederivatives used as immunomodulatory agents in vivo.

Despite various in vitro models are used for the discovery of newimmunosuppressive drugs, there are essentially no ideal animal modelsthat are simple, fast, and cost-effective. Models of organtransplantation (e.g. hearts, kidney and skin) in rodents are limited bytechnical difficulty, and cannot be used for primary screening of alarge numbers of candidate drugs. Although recently a mouse model ofdrug-mediated immunosuppression based on rejection of an allogeneicsubcutaneous tumor was reported, several significant limitations shouldbe considered. For example first, the host immune responses against thisspecific tumor seems to be T cell-dependent, and thus the activity of adrug on other cell types such as B cells, monocytes/macrophages anddentritic cells cannot be evaluated by using this model. Secondly,immunosuppressive drugs with anti-proliferation activity (e.g.rapamycin) may play a direct role on tumor angiogenesis and tumor cellgrowth. Thirdly, the feature of chronic rejection of the tumor (e.g.after 14 days) prevents from evaluating the pharmacodynamic propertiesof drugs. Finally, this animal tumor rejection model is inaccurate (e.g.quantified by size measurement) and time-costly (at least 14 days).

FACScan-based in vitro WBA has been used to investigate thepharmaco-dynamic effects of immunomodulatory drugs administrated invivo, but only by taking blood from treated recipients and stimulatingtheir lymphocytes in vitro. These methods show remarkable advantages ascompared to the purified peripheral blood mononuclear cell (PBMC)cultures in terms of maintaining cell viability, as well as the effectswithin the whole blood of immunomodulatory drugs previouslyadministrated in vivo. However, in vitro WBA has important flaws. First,the evaluation of the drug effects uses an in vitro activation system,including manipulation, dilution and stimulation of the blood, andsubsequent culture of the blood in the presence of an exogenous serum(e.g. foetal calf serum). This in vitro activation system may notnecessarily and accurately reflect the effects of drugs on leukocyteactivation in vivo, nor does this assay give information on theabsorption or metabolite production of drugs that may occur in vivo.Secondly, in vitro stimulation of the whole blood by mitogens (e.g. ConA, PHA or LPS) causes aggregation of activated leukocytes, platelets andred blood cells, as well as generation of spontaneous fluorescence by invitro damaged cells. These alterations frequently interfere with theFACS analysis. Thirdly, plastic adhesion of monocytes/macrophages duringin vitro culture may induce additional steps in order to examine thesecell types. Finally, in vitro culture of leucocytes is expensive, timeconsuming, and poorly reproducible. The above described in vivo WBAassay is able to overcome the above mentioned problems and may becharacterized as follows:

-   -   leukocyte stimulation is performed in vivo (e.g. by in vivo        injection of leucocyte stimulating agents such as, but not        limited to, ConA or LPS, thus enabling the full in vivo        evaluation of the effect of drugs on leucocyte activation in        physiological conditions,    -   various stimuli can be used that are specific for different cell        types, including T cells (e.g. ConA, anti-CD3 antibodies and        alloantigens), B cells (e.g. T cell-independent xenoantigens,        sheep red blood cells, TNP-ficoll, TNP-BA, anti-IgM antibodies        and LPS) and monocytes/macrophages (e.g. LPS, TNF-α and IFN-γ).        Given the time-dependent nature of the expression of cell        surface antigens, the effects of drugs can be studied on early        as well as on late leucocyte activation, e.g. by analyzing the        expression of early (e.g. CD69 and CD71) or late (e.g. IL-2R and        CD134) appearing antigens,    -   by varying the time of administration of a drug prior to or        after in vivo injection of the leucocyte stimuli, the        pharmacodynamic profile of the said drug can be determined.        Similarly, the effects and the duration of activity of a drug        via different routes of administration, e.g. intraperitoneously,        intramuscularly and orally, can be studied,    -   by using the same test in immunodeficient animals (e.g. T        cell-deficient nude mice), the effect of a drug on selected        immune phenomena can be investigated (e.g. on in vivo T        cell-independent B cell activation in nude mice; T cell        activation in T cell-reconstituted SCID mice that have        congenital defect on both T and B cells),    -   peripheral leukocytes from different sources, such as spleen,        lymph nodes and peritoneal cavity, can be tested,    -   in combining FACScan-based intracellular phenotype analysis with        e.g. intracellular cytokine staining, or DNA synthesis by BrdU        or 5- and 6-carboxyfluorescein diacetate succinimidyl ester        (CFSE) incorporation, or cell cycle studies, the information        obtained can be further broadened, and    -   the method is able to help in elucidating the mechanism        underlying in vivo upregulation of expression of certain        activation molecules, such as CD69, CD25, CD71, CD134 and        B7-1/2, in various types of peripheral blood leucocytes.

EXAMPLE 66 Synthesis of 4,5,6-triamino-2-methylmercaptopyrimidinedihydro-chloride

To a suspension of a 2-methylmercapto-4,5,6-triamino-pyrimidine sulfate(44.3 mmole), which may be prepared and characterised for instance asdisclosed by Taylor et al. in J. Am. Chem. Soc. (1952) 74:1644-1647, inwater (135 ml) at 80° C. was added dropwise a solution of bariumchloride dihydrate (39.8 mmole) in water (25 ml). The suspension wasstirred for 30 minutes at 80° C. The reaction mixture was cooled downand barium sulfate was filtered off over Celite. The filtrate wasevaporated in vacuo and co-evaporated with toluene yielding the titlecompound as a yellow powder (10.2 g, 94% yield).

EXAMPLE 67 Synthesis of4-amino-2-methylmercapto-6-(3,4-dimethoxy-phenyl)pteridine

To a suspension of 4,5,6-triamino-2-methylmercaptopyrimidinedihydrochloride (7.42 mmole, 1.81 g) in methanol (20 ml) was added asolution of 3,4-dimethoxyphenylglyoxaloxime (5.94 mmole, 1.24 g) inmethanol. The resulting reaction mixture was refluxed for 3 hours. Thereaction mixture was neutralised with concentrated aqueous ammonia untilpH 9 was reached. The resulting precipitate was filtered off and furtherpurified by flash chromatography (silica, using an ethyl acetate/hexanemixture in a 4:6 ratio) yielding the pure title compound as a yellowpowder which was characterised as follows: MS: m/z (%) 330 ([M+H]⁺,100), 681 ([2M+Na]⁺, 30); UV (MeOH, nm): 292, 397.

EXAMPLE 68 Synthesis of2,4-di-(thienyl-2-methylamino)-6-(3,4-dimethoxy-Phenyl) pteridine

A solution of the compound of example 67 (100 mg, 0.304 mmole) in2-thiophenemethylamine (12 ml) was refluxed overnight. The solvents wereremoved in vacuo and the residue was purified first by flashchromatography (silica, gradient from 2:98 to 3:97 CH₃OH/CH₂Cl₂) andthen by preparative TLC (silica, EtOAc/hexane 1:1) yielding the titlecompound as a yellow powder (85 mg, 57%) which was characterised asfollows: MS: m/z (%): 491 ([M+H]⁺, 100); UV (MeOH, nm): 229, 296, 414.

EXAMPLE 69 Synthesis of 2,5,6-triamino-4-morpholino-pyrimidinedihydro-chloride

In a 10 L 4-neck flask equipped with a mechanical stirrer, a thermometerand a heating mantle was placed 2,6-diamino-4-chloropyrimidine (870.5 g;6.0 mol) In water (3190 ml). To the stirred resulting white suspensionwas added morpholine (1113 g; 12.8 moles) in one portion. Thetemperature raised from 14 to 28° C. and the mixture was heated until at75° C. a clear solution was obtained and refluxed for 16 hours. Aftercomplete conversion of the starting material (controlled by HPLCanalysis) the reaction mixture was cooled to room temperature and NaNO₂(465 g, 6.74 mole) was added in three portions. No heat effect wasobserved but the product precipitated out of the mixture. Duringsubsequent dropwise addition of acetic acid (600 g, 9.0 moles) themixture became so thick that faster stirring and additional water (850mL) were necessary to achieve a well mixable suspension. A temperatureraise from 15 to 28° C. was measured. After stirring for 2 hours, HPLCanalysis showed complete conversion. The mixture was then allowed tocool down to room temperature and transferred to a 20 L flask. To theadd (pH 4) purple slurry was added water (3500 mL) and then an aqueousNaOH solution (165 g in 825 mL) in order to neutralise to pH 7. Thesuspension was cooled down to 10-12° C. in an ice-water bath and Na₂S₂O₄(4.68 kg, 26.9 moles) was added in portions of about 500 g at 5 minutesintervals. A temperature raise to 30° C. was observed. The mixture wasleft stirring overnight at room temperature before being analysed (HPLC:99.3% conversion). The suspension was filtered over filter cloth and thewet cake was washed with 2.77 L water leaving 1.5 kg of wet materialwhich was slurried in isopropanol (10.2 L) and to the suspension wasadded dropwise 36% HCl/H₂O (1.16 L) during which the temperature raisedfrom 15 to 30° C. Subsequent heating of the slurry to 40° C. afforded analmost clear solution and crystallisation started suddenly showing anexotherm to 50° C. The suspension was cooled down to room temperaturebefore the suspension was filtered over filter cloth. The wet cake wasagain slurried in isopropanol (5.0 L), again filtered over filter clothand dried at 40° C. in vacuo (200 mbar) for several days. According tothis procedure was obtained 1.2 kg (yield 54.1%) of2,5,6-triamino-4-morpholino-pyrimidine dihydrochloride with a purityabove 99.9% and characterised as follows: UV (MeOH, nm): 225, 254.

EXAMPLE 70 Synthesis of 4-acetamidophenylplyoxalmonoxime

SeO₂ (34.18 g, 0.308 mole) and 4-acetamidoacetophenone (49.62 g, 0.28mole) were suspended in a mixture of dioxane (250 ml) and water (10 mland the solution was heated to 100° C. for 16 hours. The hot solutionwas filtered on a paper filter and the filtrate was evaporated todryness. The oily residue was then partitioned between CHCl₃ (400 ml)and a saturated solution of sodium hydrogen-carbonate (200 ml). Aprecipitation occurs In the aqueous layer. The organic phase wascollected and the aqueous layer was filtered over a fritted glass. Thesolid (4-acetamidoglyoxaldehyde) was washed with water and kept for thenext step. The aqueous layer was extracted several times with CHCl₃ andall fractions were collected and evaporated to dryness. The oily residueand the precipitate were put together and suspended in a mixture ofwater (240 ml) and MeOH (60 ml). Then acetonoxime (20.46 g, 0.28 mole)was added and the pH was adjusted to a range of 3-4 with 1 N HCl. Thesolution was heated to 70° C. for 1 hour, then cooled to 0° C. and theresulting crystals collected. After washing with cold water, thendiethylether, drying in a vacuum dessicator over P₂O₅ afforded 33.6 g ofthe desired compound (yield 58%) as yellowish crystals with a purity of95%, which were characterised as follows: MS: m/z (%): 207 ([M+H]⁺,100); UV (MeOH, nm): 241.6, 278.3.

EXAMPLE 71 Synthesis of 3-acetamidophenylglyoxalmonoxime

SeO₂ (36.62 g, 0.33 mole) and 3-acetamidoacetophenone (53.16 g, 0.30mole) were suspended in a mixture of dioxane (250 ml) and water (10 ml)and the solution was heated to 100° C. for 16 hours. The hot solutionwas filtered on a paper filter and the filtrate was evaporated todryness. The oily residue was then suspended in a mixture of water (240ml) and MeOH (60 ml) and acetonoxime (21.93 g, 0.30 mol) was added andthe pH was adjusted to a range of 3-4 with 6 N HCl. The solution washeated to 70° C. for 2 hours and after cooling, the brownish precipitatewas filtered and re-crystallised from toluene to afford 45.0 g of thedesired compound (yield 72%) with a purity of 98%, which wascharacterised as follows: MS: m/z (%): 207 ([M+H]⁺, 100), 229 ([M+Na]⁺,80); UV (MeOH, nm): 239.2.

EXAMPLE 72 Synthesis of 2-amino-4-morpholino-6-(4-acetanilide)pteridine

2,4,5-triamino-6-morpholinopyrimidine dihydrochloride (14.1 g, 50 mmole)was refluxed in MeOH (300 ml) and the compound of example 70 (11.33 g,55 mmole) in MeOH (150 ml) was added. The mixture was refluxed for 3hours and after cooling, the yellowish precipitate was filtered, washedwith water, methanol, ether and dried at 110° C. for 3 hours to afford15.7 g of the desired product (yield 86%) which, afterre-crystallisation from DMF/H₂O, was obtained with a purity of 97% andcharacterised as follows: MS: m/z (%): 366 ([M+H]⁺, 100), 753 ([2M+Na]⁺,15); UV (MeOH, nm): 213, 307, 408.

EXAMPLE 73 Synthesis of 2-amino-4-morpholino-6-(3-acetanilide)pteridine

Repeating the procedure of example 72 but starting from the compound ofexample 71, the title pteridine derivative (16.1 g) was obtained with ayield of 88% and a purity of 97%, and characterised as follows: MS: m/z(%): 366 ([M+H]⁺, 100), 753 ([2M+Na]⁺, 80); UV (MeOH, nm): 220, 294,402.

EXAMPLE 74 Synthesis of 2-amino-morpholino-6-(4-aminophenyl)pteridine

The crude compound of example 72 (20 mmole) was refluxed in a mixture ofMeOH/HCl 6N-1/1 (400 ml) for 2 hours and cooled in an ice bath. Theremaining solid was filtered and the pH was adjusted to 10 with NaOH(10N). The orange precipitate was filtered, washed with water, ethanol,ether and dried in a vacuum dessicator over P₂O₅ to afford 4.7 g (yield:73%) of the desired compound as an orange solid with a purity of 98%,which was characterised as follows: MS: m/z (%): 324 ([M+H]⁺, 100); UV(MeOH, nm): 216, 316, 422; ¹H NMR (200 MHz, in DMSO-d6, ppm): 3.78 (brs, 4H); 4.33 (br s, 4H); 5.54 (br s, 2H); 6.63 (br s, 2H); 6.67 (d, 2H,J=8.4 Hz); 7.75 (d, 2H, J=8.4 Hz); 9.15 (s, 1H).

EXAMPLE 75 Synthesis of 2-amino-4-morpholino-6-(3-aminophenyl)pteridine

Repeating the procedure of example 74 but starting from the compound ofexample 73, the title pteridine derivative (4.88 g) was obtained with ayield of 74% and a purity of 99.34%, and characterised as follows: MS:m/z (%): 324 ([M+H]⁺, 100); UV (MeOH, nm): 218, 292, 403.

EXAMPLES 76 TO 82 Coupling Reaction Between a2-amino-4-morpholino-6-(4-amino-phenyl)pteridine and a Carboxylic AcidChloride, Sulfonyl Chloride or Carbamoyl Chloride

The compound of example 74 (162 mg, 0.5 mmole) was suspended in dioxane(10 ml) and triethylamine was added (84 μl, 0.6 mmole). Then a suitablecarboxylic acid chloride or sulfonyl chloride or carbamoyl chloride(0.55 mmole) was added and the mixture was stirred at room temperatureuntil completion of the reaction. After evaporation to dryness, thecrude resulting product was purified by flash chromatography on silicagel using a gradient of MeOH (1-5%) in dichloromethane. Yields variedfrom 50 to 80%. Using this procedure, the following pteridinederivatives were prepared:

-   2-amino-4-morpholino-6-(4-benzoylaminophenyl)pteridine (example 76),    purity 98.94%, characterised as follows: MS: m/z (%): 428 ([M+H]⁺,    100), 877 [2M+Na]⁺, 40); UV (MeOH, m): 313, 408;-   2-amino-4-morpholino-6-(4-phenoxyacetylaminophenyl)pteridine    (example 77), purity 97.18%, characterised as follows: MS: m/z (%):    458 ([M+H]⁺, 100); UV (MeOH, nm): 305, 407;-   2-amino-4-morpholino-6-(4-propionylaminophenyl)pteridine (example    78), purity 98.86%, characterised as follows: MS: m/z (%): 380    ([M+H]⁺, 100), 781 ([2M+Na]⁺, 20); UV (MeOH, nm): 213, 307, 408;-   2-amino-4-morpholino-6-(4-furoylaminophenyl)pteridine (example 79),    purity 93.96%, characterised as follows: MS: m/z (%): 418 ([M+H]⁺,    100); UV (MeOH, nm): 213, 316, 408;-   2-amino-4-morpholino-6-(4-cyclohexanoylaminophenyl)pteridine    (example 80), purity 96.91%, characterised as follows: MS: m/z (%):    434 ([M+H]⁺, 100), 889 ([2M+Na]⁺, 10); UV (MeOH, nm): 308, 408;-   2-amino-4-morpholino-6-[4-(4-chlorobenzoyl)aminophenyl]pteridine    (example 81), purity 96.48%, characterised as follows: MS: m/z (%):    462 ([M+H]⁺, 100); UV (MeOH, nm): 313.9, 407; and-   2-amino-4-morpholino-6-(4-benzyloxyacetylaminophenyl)pteridine    (example 82), purity 98.45%, characterised as follows: MS: m/z (%):    472 ([M+H]⁺, 100), 942 ([2M+H]⁺, 5), 965 ([2M+Na]⁺, 10); UV (MeOH,    nm): 307, 407.

EXAMPLES 83 TO 97 Coupling Reaction Between a2-amino-4-morpholino-6-aminophenyl pteridine and a Carboxylic AcidChloride, Sulfonyl Chloride or Carbamoyl Chloride

The compound of example 74 or example 75 (162 mg, 0.5 mmole) wassuspended in pyridine (10 ml). Then a suitable carboxylic acid chlorideor sulfonyl chloride or carbamoyl chloride (0.55 mmole) was added andthe mixture was stirred at room temperature until completion of thereaction. After evaporation to dryness, the crude resulting product waspurified by flash chromatography on silica gel using a gradient of MeOH(1-5%) in dichloromethane. Yields varied from 20 to 80%. Using thisprocedure, the following pteridine derivatives were prepared:

-   2-amino-4-morpholino-6-(4-isonicotinoylaminophenyl)pteridine    (example 83) was characterised as follows: MS: m/z (%): 429 ([M+H]⁺,    100), 879 (2M+Na]⁺, 5); UV (MeOH, m): 213, 313, 407; purity 95.01%;-   2-amino-4-morpholino-6-(4-naphtoylaminophenyl)pteridine (example 84)    was characterised as follows: MS: m/z (%): 478 ([M+H]⁺, 100), 977    ([2M+Na]⁺, 5); UV (MeOH, nm): 213, 313, 407; purity 95.01%;-   2-amino-4-morpholino-6-(4-methylsulfonylaminophenyl)pteridine    (example 85) was characterised as follows: MS: m/z (%): 502 ([M+H]⁺,    100); UV (MeOH, nm): 301, 422; purity 96.81%;-   2-amino-4-morpholino-6-(4-ethylsuccinylaminophenyl)pteridine    (example 86) was characterised as follows: MS: m/z (%): 452 ([M+H]⁺,    100), 925 ([2M+Na]⁺, 15); UV (MeOH, nm): 213, 308, 408; purity:    98.98%;-   2-amino-4-morpholino-6-[4-(4-methylbenzoate)aminophenyl)pteridine    (example 87) was characterised as follows: MS: m/z (%): 486 ([M+H]⁺,    100); UV (MeOH, nm): 236, 315, 407; purity 99.57%;-   2-amino-4-morpholino-6-(3-benzoylaminophenyl)pteridine (example 88)    was characterised as follows: MS: m/z (%): 428 ([M+H]⁺, 100); UV    (MeOH, nm): 216, 293, 402; purity: 96.46%;-   2-amino-4-morpholino-6-(3-benzensulfonylaminophenyl)pteridine    (example 89) was characterised as follows: MS: m/z (%): 464 ([M+H]⁺,    100); UV (MeOH, nm): 216, 295, 402; purity: 97.49%;-   2-amino-4-morpholino-6-(3-phenoxyacetylaminophenyl)pteridine    (example 90) was characterised as follows: MS: m/z (%): 458 ([M+H]⁺,    100); UV (MeOH, nm): 219, 294, 402; purity 98.96%;-   2-amino-4-morpholino-6-(3-isonicotinoylaminophenyl)pteridine    (example 91) was characterised as follows: MS: m/z (%): 429 ([M+H]⁺,    100); UV (MeOH, nm): 216, 294, 402; purity 93.92%;-   2-amino-4-morpholino-6-(3-cyclohexanoylaminophenyl)pteridine    (example 92) was characterised as follows: MS: m/z (%): 434 ([M+H]⁺,    100); UV (MeOH, nm): 222, 245, 294, 402; purity 99.50%;-   2-amino-4-morpholino-6-[3-(4-methylbenzoate)aminophenyl]pteridine    (example 93) was characterised as follows: MS: m/z (%): 486 ([M+H]⁺,    100); UV (MeOH, nm): 218, 238, 294, 402; purity 97.44%;-   2-amino-4-morpholino-6-(3-ethylsuccinylaminophenyl)pteridine    (example 94) was characterised as follows: MS: m/z (%): 452 ([M+H]⁺,    100); UV (MeOH, nm): 222, 245, 294, 402; purity 94.16%;-   2-amino-4-morpholino-6-(3-ethylmalonylaminophenyl)pteridine    (example 95) was characterised as follows: MS: m/z (%): 438 ([M+H]⁺,    100); UV (MeOH, nm): 220, 244, 294, 402; purity 90.89%;-   2-amino-4-morpholino-6-(3-benzyloxyacetylaminophenyl)pteridine    (example 96) was characterised as follows: MS: m/z (%): 472 ([M+H]⁺,    100); UV (MeOH, nm): 216, 243, 294, 402; purity 99.70%; and-   2-amino-4-morpholino-6-(3-ethylsulfonylaminophenyl)pteridine    (example 97) was characterised as follows: MS: m/z (%): 4716    ([M+H]⁺, 100); UV (MeOH, nm): 218, 295, 402; purity 92.54%.

EXAMPLES 98 TO 101 Coupling Reaction Between2-amino-4-morpholino-6-(3-aminophenyl)pteridine and Various CarboxylicAcids

The compound of example 75 (323 mg, 1 mmole), a carboxylic acid (1.1mmole) and DIEA (2 mmole) were suspended in dry DMF (10 ml) andbenzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate)was added (1.1 mmole). The mixture was stirred at room temperature untilcompletion of the reaction and then diluted with water. The aqueouslayer was extracted with chloroform and the organic layer was evaporatedto dryness. The crude resulting product was purified on silica gel usinga gradient of MeOH (1-5%) in dichloromethane. Yields varied from 20 to50%. The following compounds were made according to this procedure:

-   2-amino-4-morpholino-6-[3-Boc-(L)-phenylalanine-aminophenyl]pteridine    (example 98) was characterised as follows: MS: m/z (%): 1141    ([2M+H]⁺, 10), 571 ([M+H]⁺, 100), 515 ([M-tbu+H]⁺, 50), 471    ([M-Boc+H]⁺, 10); UV (MeOH, nm): 213, 245, 294, 402; purity 96.72%;-   2-amino-4-morpholino-6-[3-Boc-(D)-phenylalanine-aminophenyl]pteridine    (example 99) was characterised as follows: MS: m/z (%): 571 ([M+H]⁺,    100), 515 ([M-tBu+H]⁺, 50), 471 ([M-Boc+H]⁺, 10); UV (MeOH, nm):    213, 245, 294, 402; purity: 95.30%;-   2-amino-4-morpholino-6-[3-Boc-(L)-tryptophane-aminophenyl]pteridine    (example 100) was characterised as follows: MS: m/z (%): 610    ([M+H]⁺, 100), 554 ([M-tBu+H]⁺, 50), 510 ([M-Boc+H]⁺, 10); UV (MeOH,    nm): 220, 291, 402; purity 91.74%; and-   2-amino-4-morpholino-6-[3-Boc-(D)-tryptophane-aminophenyl]pteridine    (example 101) was characterised as follows: MS: m/z (%): 610    ([M+H]⁺, 100), 554 (M-tBu+H]⁺, 50), 510 ([M-Boc+H]⁺, 10); UV (MeOH,    nm): 220, 291, 402; purity 84.25%.

EXAMPLE 102 Synthesis of2-amino-4-morpholino-6-(4-hydroxyphenyl)pteridine

To a solution of 2,5,6-triamino-4-morpholino-pyrimidine dihydrochloridesalt (5.05 g, 17.8 mmole) in methanol (120 ml) was added4-hydroxyphenylglyoxalmonoxime (2.95 g, 17.8 mmole). The reactionmixture was refluxed for 3 hours and then cooled down to roomtemperature. The yellow precipitate was filtered off, washed with wateryielding the title compound as a chromatographically pure yellow powderwhich was further characterised as follows: MS: m/z (%): 671 ([2M+Na]⁺,5), 325 ([M+H]⁺, 100); UV (MeOH, nm): 213, 302, 411.

EXAMPLES 103 TO 111 Synthesis of2-amino-4-morpholino-6-(4-alkoxy-phenyl)pteridines

To a solution of the compound of example 102 (0.65 mmole, 210 mg) in DMFwas added K₂CO₃ (0.78 mmole, 108 mg) and an appropriate alkyl halide(0.78 mmole). The reaction was stirred overnight at room temperature.The reaction was quenched with water and extracted with CH₂Cl₂. Theorganic phase was evaporated in vacuo and the residue purified by flashchromatography (silica, gradient from 1:99 to 3:97 CH₃OH/CH₂Cl₂)yielding the title compound as a yellow powder in yields ranging from 15to 55%. The following compounds were made using this procedure:

-   2-amino-4-morpholino-6-(4-ethoxyphenyl)pteridine (example 103) was    obtained from iodoethane and characterised as follows: MS: m/z (%):    727 ([2M+Na]⁺, 5), 353 ([M+H]⁺, 100); UV (MeOH, nm): 211, 302, 410;-   2-amino-4-morpholino-6-(4-benzyloxyphenyl)pteridine (example 104)    was obtained from benzyl bromide and characterised as follows: MS:    m/z (%): 727 ([2M+Na]⁺, 5), 353 ([M+H]⁺, 100); UV (MeOH, nm): 245,    302, 410;-   2-amino-4-morpholino-6-(4-(phenethyloxy)-phenyl)pteridine    (example 105) was obtained from phenylethyl bromide and    characterised as follows: MS: m/z (%): 428 ([M+H]⁺, 100); UV (MeOH,    nm): 245, 303, 410;-   2-amino-4-morpholino-6-(4-phenoxy-butyronitrile) pteridine    (example 106) was obtained from 4-bromobutyronitrile and    characterised as follows: MS: m/z (%): 391 ([M+H]⁺, 100);-   2-amino-4-morpholino-6-(4-propoxy-phenyl)pteridine (example 107) was    obtained from propyl iodide and characterised as follows: MS: m/z    (%): 367 ([M+H]⁺, 100);-   2-amino-4-morpholino-6-(4-phenoxy-butyric acid ethyl ester)    pteridine (example 108) was obtained from ethyl-4-bromobutyrate and    characterised as follows: MS: m/z (%): 439 ([M+H]⁺, 100);-   2-amino-4-morpholino-6-(4-phenoxy-acetic acid ethyl ester) pteridine    (example 109) was obtained from ethyl bromoacetate and characterised    as follows: MS: m/z (%): 843 ([2M+H]⁺, 100), 411 ([M+H]⁺, 100;-   2-amino-4-morpholino-6-(4-(2-methoxyethoxy)-phenyl)pteridine    (example 110) was obtained from 2-bromoethylmethylether and    characterised as follows: MS: m/z (%): 787 ([2M+H]⁺, 25), 383    ([M+H]⁺, 100); and-   2-amino-4-morpholino-6-(4-butoxy-phenyl)-pteridine (example 111) was    obtained from bromobutane and characterised as follows: MS: m/z (%):    381 ([M+H]⁺, 100).

EXAMPLES 112 TO 120 Synthesis of 2-amino-4-alkylamino-6-aryl-pteridinesand 2-amino-4-arylalkylamino-6-aryl-pteridines

To a suspension of2-acetylamino-4-(1,2,4-triazolyl)-6-[3,4-(dimethoxyphenyl)]pteridine(0.5 mmole) in dioxane (5 ml) was added the desired alkylamine orarylalkylamine (1 mmole). The suspension was stirred at room temperaturefor 16 hours. The solvent was evaporated in vacuo yielding crude2-acetylamino-4-alkylamino-6-[3,4-(dimethoxyphenyl)]pteridine or2-acetylamino-4-arylalkylamino-6-[3,4-(dimethoxyphenyl)]pteridine.Deprotection of the acetyl group was achieved by dissolving the cruderesidue in a mixture of CH₃OH/20% K₂CO₃ in water (1:1). The solution wasstirred at room temperature for 16 hours. Evaporation of the solvents invacuo, followed by purification of the residue by preparative TLC(silica, using a CH₃OH/CH₂Cl₂ mixture (5:95) as the eluent) afforded thedesired compound as a yellow powder in yields ranging from 30 to 65%,depending upon the starting alkylamine or arylalkylamine.

The following compounds were synthesized according to this procedure andcharacterized as follows:

-   2-amino-4-(diethanolamino)-6-[[3,4-(dimethoxyphenyl)]pteridine    (example 112) synthesized from diethanolamine; MS: m/z (%): 387    ([M+H]⁺, 100);-   2-amino-4-(benzylamino)-6-[[(3,4-(dimethoxyphenyl)]pteridine    (example 113) synthesized from benzylamine; MS: m/z (%): 389    ([M+H]⁺, 100);-   2-amino-4-(phenylethylamino)-6-[[3,4-(dimethoxyphenyl)]pteridine    (example 114) synthesized from phenethylamine; MS: m/z (%): 403    ([M+H]⁺, 100);-   2-amino-4-(4-methyl-piperidine)-6-[[3,4-(dimethoxyphenyl)]pteridine    (example 115) synthesized from 4-methyl-piperidine; MS: m/z (%): 381    ([M+H]⁺, 100);-   2-amino-4-(2-thienylmethylamino)-6-[[3,4-(dimethoxyphenyl)]pteridine    (example 116) synthesized from 2-thienylamine; MS: m/z (%): 395    ([M+H]⁺, 100);-   2-amino-4-(1,2,3,6-tetrahydropyridino)-6-[[3,4-(dimethoxyphenyl)]pteridine    (example 117) synthesized from 1,2,3,6-tetrahydropyridine; MS: m/z    (%): 365 ([M+H]⁺, 100);-   2-amino-4-thiomorpholine-6-[[3,4-(dimethoxyphenyl)]pteridine    (example 118) synthesized from thiomorpholine; MS: m/z (%): 385    ([M+H]⁺, 100);-   2-amino-4-((R)-sec-butylamine)-6-[[3,4-(dimethoxyphenyl)]pteridine    (example 119) synthesized from (R)-sec-butylamine; MS: m/z (%): 355    ([M+H]⁺, 100); and-   2-amino-4-((S)-sec-butylamine)-6-[[3,4-(dimethoxyphenyl)]pteridine    (example 120) synthesized from (S)-sec-butylamine; MS: m/z (%): 355    ([M+H]⁺, 100).

EXAMPLE 121 Mixed Lymphocyte Reaction Assay

Pteridine derivatives were first dissolved (10 mM) in dimethylsulfoxide(hereinafter referred as DMSO) and further diluted in culture mediumbefore use for the following in vitro experiments. The commerciallyavailable culture medium consisted of RPMI-1640+10% foetal calf serum(FCS). Some pteridine derivatives described in the previous examples (asindicated in table 3 below) were tested in the following mixedlymphocyte reaction (MLR) assay.

Peripheral blood mononuclear cells (hereinafter referred as PBMC) wereisolated from heparinized peripheral blood by density gradientcentrifugation over Lymphoprep (Nycomed, Maorstua, Norway). AllogeneicPBMC or Eppstein-Barr Virus-transformed human B cells [commerciallyavailable under the trade name RPMI1788 (ATCC name CCL156)]whichstrongly express B7-1 and B7-2 antigens were used as stimulator cellsafter irradiation with 30 Gy. MLR was performed in triplicate wells.After 5 days incubation at 37° C., 1 μCi [³H]-thymidine was added toeach cup. After a further 16 hours incubation, cells were harvested andcounted in a β-counter. Inhibition of proliferation by a compound (drug)described in some of the previous examples was counted using theformula:

${\%\mspace{11mu}{inhibition}} = {\frac{\left( {{cpm} + {drugs}} \right) - \left( {{cpm}\mspace{14mu}{{cult}.\mspace{14mu}{med}}} \right)}{\left( {{cpm} - {drugs}} \right) - \left( {{OD}\mspace{14mu}{{cult}.\mspace{14mu}{med}}} \right)} \times 100}$wherein cpm is the thymidine count per minute. The MLR assay is regardedby those skilled in the art as an in vitro analogue of the transplantrejection since it is based on the recognition of allogeneic majorhistocompatibility antigens on the stimulator leukocytes, by respondinglymphocytes.

Table 3 below shows the IC₅₀ values for various pteridine derivatives ofthis invention in the MLR test. The IC₅₀ value represents the lowestconcentration of said pteridine derivative (expressed in μmole/l) thatresulted in a 50% suppression of the MLR.

TABLE 3 Example n° MLR Example n° MLR Example n° MLR 68 0.8 72 8.6 736.4 74 1.1 75 4.1 76 2.1 77 2.5 78 6.5 79 7.8 80 4.1 83 3.9 84 4.0 884.9 89 0.8 90 10.0 91 10.0 92 2.9 96 4.1 97 3.3 98 3.9 99 4.1 100 0.9101 0.6 102 4.3 103 3.7 107 6.8 110 5.4 112 6.0 114 6.0 115 0.8 116 4.1117 0.9 118 0.4 119 0.5 120 0.2

EXAMPLE 122 TNF-α Assay

Peripheral blood mononuclear cells (herein referred as PBMC), inresponse to stimulation by lipopolysaccharide (hereinafter LPS), agram-negative bacterial endotoxin, produce various chemokines, inparticular human TNF-α. Inhibition of the activation of PBMC cantherefore be measured by the level of suppression of the production ofTNF-α by PBMC in response to stimulation by LPS.

Such inhibition measurement was performed as follows: PBMC were isolatedfrom heparinized peripheral blood by density gradient centrifugationover Lymphoprep (commercially available from Nycomed, Norway). LPS wasthen added to the PMBC suspension in complete medium (10⁶ cells/ml) at afinal concentration of 1 μg/ml. The pteridine derivative to be testedwas added at different concentrations (0.1 μM, 1 μM and 10 μM) and thecells were incubated at 37° C. for 72 hours in 5% CO₂. The supernatantswere collected, then TNF-α concentrations were measured with ananti-TNF-α antibody in a sandwich ELISA (Duo Set ELISA human TNF-α,commercially available from R&D Systems, United Kingdom). Thecolorimetric reading of the ELISA was measured by a Multiskan RC platereader (commercially available from ThermoLabsystems, Finland) at 450 nm(reference wavelength: 690 nm). Data analysis was performed with Ascentsoftware 2.6. (also from ThermoLabsystems, Finland): a standard curve(recombinant human TNFα) was drawn and the amount (pg/ml) of each sampleon the standard curve was determined.

The % suppression of human TNFα production by the pteridine derivativesof the invention (drugs) was calculated using the formula:

${\%\mspace{11mu}{suppression}} = \frac{\begin{matrix}{{{pg}\text{/}{ml}\mspace{14mu}{in}\mspace{14mu}{drugs}} -} \\{{pg}\text{/}{ml}\mspace{14mu}{in}\mspace{14mu}{{cult}.\mspace{11mu}{med}.}}\end{matrix}}{\begin{matrix}{\left( {{pg}\text{/}{ml}\mspace{14mu}{in}\mspace{14mu}{{cult}.\mspace{14mu}{med}.{+ {LPS}}}} \right) -} \\{{pg}\text{/}{ml}\mspace{14mu}{{cult}.\mspace{14mu}{med}.}}\end{matrix}}$

Table 4 below shows the IC₅₀ values (expressed in μM) of the testedpteridine derivatives in the TNF-α assay.

TABLE 4 Example n° TNF-α Example n° TNF-α Example n° TNF-α 72 8.4 74 2.577 4.9 83 4.4 88 4.3 89 0.5 91 7.3 92 5.0 94 5.5 96 7.7 97 10.0 98 2.9102 2.1 103 10.0 107 10.0 108 0.9 109 0.6 110 7.2 112 3.4 113 6.5 1147.1 115 0.3 116 7.2 117 0.5 118 0.5 119 0.2 120 0.3

1. A pteridine derivative selected from the group consisting of:2-amino-4-morpholino-6-(4-acetanilide)pteridine,2-amino-4-morpholino-6-(3-acetanilide)pteridine,2-amino-4-morpholino-6-(4-aminophenyl)pteridine,2-amino-4-morpholino-6-(3-aminophenyl)pteridine,2-amino-4-morpholino-6-(4-benzoylaminophenyl)pteridine,2-amino-4-morpholino-6-(4-phenoxyacetylaminophenyl)pteridine,2-amino-4-morpholino-6-(4-propionylaminophenyl)pteridine,2-amino-4-morpholino-6-(4-furoylaminophenyl)pteridine,2-amino-4-morpholino-6-(4-cyclohexanoylaminophenyl)pteridine,2-amino-4-morpholino-6-[4-(4-chlorobenzoyl)aminophenyl]pteridine,2-amino-4-morpholino-6-(4-benzyloxyacetylaminophenyl)pteridine,2-amino-4-morpholino-6-(4-isonicotinoylaminophenyl)pteridine;2-amino-4-morpholino-6-(4-naphtoylaminophenyl)pteridine;2-amino-4-morpholino-6-(4-methylsulfonylaminophenyl)pteridine;2-amino-4-morpholino-6-(4-ethylsuccinylaminophenyl)pteridine;2-amino-4-morpholino-6-[4-(4-methylbenzoate)aminophenyl)pteridine;2-amino-4-morpholino-6-(3-benzoylaminophenyl)pteridine;2-amino-4-morpholino-6-(3-benzensulfonylaminophenyl)pteridine,2-amino-4-morpholino-6-(3-phenoxyacetylaminophenyl)pteridine;2-amino-4-morpholino-6-(3-isonicotinoylaminophenyl)pteridine;2-amino-4-morpholino-6-(3-cyclohexanoylaminophenyl)pteridine;2-amino-4-morpholino-6-[3-(4-methylbenzoate)aminophenyl]pteridine;2-amino-4-morpholino-6-(3-ethylsuccinylaminophenyl)pteridine;2-amino-4-morpholino-6-(3-ethylmalonylaminophenyl)pteridine;2-amino-4-morpholino-6-(3-benzyloxyacetylaminophenyl)pteridine,2-amino-4-morpholino-6-(3-ethylsulfonylaminophenyl)pteridine,2-amino-4-morpholino-6-[3-Boc-(L)-phenylalanine-aminophenyl]pteridine;2-amino-4-morpholino-6-[3-Boc-(D)-phenylalanine-aminophenyl]pteridine;2-amino-4-morpholino-6-[3-Boc-(L)-tryptophane-aminophenyl]pteridine;2-amino-4-morpholino-6-[3-Boc-(D)-tryptophane-aminophenyl]pteridine,2-amino-4-morpholino-6-(4-hydroxyphenyl)pteridine,2,4-di-(thienyl-2-methylarnino)-6-(3,4-dimethoxy-phenyl)pteridine,2-amino-4-morpholino-6-(4-benzyloxyphenyl)pteridine,2-amino-4-morpholino-6-(4-(phenethyloxy)-phenyl)pteridine,2-amino-4-morpholino-6-(4-phenoxy-butyronitrile)pteridine,2-amino-4-morpholino-6-(4-propoxy-phenyl)pteridine,2-amino-4-morpholino-6-(4-phenoxy-butyric acid ethyl ester)pteridine,2-amino-4-morpholino-6-(4-phenoxy-acetic acid ethyl ester)pteridine,2-amino-4-morpholino-6-(4-(2-methoxyethoxy)-phenyl)pteridine,2-amino-4-morpholino-6-(4-butoxy-phenyl)-pteridine,2-amino-4-(diethanolamino)-6-[[3,4-(dimethoxyphenyl)]pteridine,2-amino-4-(benzylamino)-6-[[3,4-(dimethoxyphenyl)]pteridine;2-amino-4-(phenylethylamino)-6-[[3,4-(dimethoxyphenyl)]pteridine,2-amino-4-(2-thienylmethylamino)-6-[[3,4-(dimethoxyphenyl)]pteridine,2-amino-4-thiomorpholine-6-[[3,4-(dimethoxyphenyl)]pteridine.
 2. Apharmaceutical composition comprising as an active principle at leastone pteridine derivative according to claim
 1. 3. A pharmaceuticalcomposition comprising as an active principle at least one pteridinederivative according to claim 1, and further comprising one or morebiologically active drugs selected from the group consisting ofimmuno-suppressant and/or immunomodulator drugs.
 4. A pharmaceuticalcomposition comprising as an active principle at least one pteridinederivative according to claim 1, and further comprising one or moreimmunosuppressant drugs selected from the group consisting ofcyclosporin A; pentoxyfylline; daltroban, sirolimus, tacrolimus;rapamycin; leflunomide; mycophenolic acid and salts thereof;azathioprine, brequinar; gusperimus; 6-mercaptopurine; mizoribine;chloroquine; hydroxy-chloroquine; etanercept; infliximab; and kineret.5. A pharmaceutical composition comprising as an active principle atleast onepteridine derivative according to claim 1 and furthercomprising one or more immunomodulator drugs selected from the groupconsisting of acemannan, amiprilose, bucillamine, ditiocarb sodium,imiquimod, Inosine Pranobex, interferon-β, interferon-γ, lentinan,levamisole, pidotimod, romurtide, platonin, procodazole, propagermanium,thymomodulin, thymopentin and ubenimex.
 6. A pharmaceutical compositioncomprising as an active principle at least one pteridine derivativeaccording to claim 1 and further comprising one or more pharmaceuticallyacceptable carriers or excipients.